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Immune-reactivity of recombinant isoforms of the major house dust mite allergen Der p 2
G A Hakkaart1, M D Chapman, R C Aalberse
1Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, University of Amsterdam.
Background:
Recombinant Der p 2, expressed in yeast, lacked reactivity with 5 monoclonal antibodies against natural Der p 2.
Objective:
The aim of this study was to investigate whether the lack of reactivity with recombinant Der p 2 can be explained by the existence of isoforms.
Methods:
By site-directed mutagenesis three recombinant isoforms of Der p 2 were produced. Reactivity with monoclonal antibodies and human IgE was analysed by means of RAST and RAST-inhibition.
Results:
All five monoclonals that lacked reactivity with the originally selected isoform, showed reactivity upon replacement of aspartic acid by asparagine at position 114. The other two substitutions (at position 26 and 47) had no effect. Binding of human IgE (n = 10) was not significantly influenced by the isogenetic variation at position 114.
Conclusions:
Monoclonal antibodies raised against natural Der p 2 can sometimes discriminate between different isoforms, allowing the study of the natural occurrence of isoforms. For application in allergen-measurement assays, non-discriminating monoclonal antibodies should be selected.
Insights
Recombinant Der p 2 isoforms were created to understand why some antibodies didn't react. A change at position 114 restored antibody binding, suggesting isoforms affect antibody recognition in Der p 2 allergen studies.
Area of Science:
- Molecular biology
- Immunology
- Allergen research
Background:
- Recombinant Der p 2 produced in yeast showed limited reactivity with monoclonal antibodies specific to natural Der p 2.
- This discrepancy prompted an investigation into potential causes, such as the existence of Der p 2 isoforms.
Purpose of the Study:
- To investigate if the lack of reactivity of certain monoclonal antibodies with recombinant Der p 2 is due to different isoforms.
- To characterize the impact of specific amino acid substitutions on Der p 2 isoform recognition.
Main Methods:
- Site-directed mutagenesis was used to create three recombinant Der p 2 isoforms.
- Reactivity of these isoforms with monoclonal antibodies and human IgE was assessed using Radioallergosorbent Test (RAST) and RAST-inhibition assays.
Main Results:
- Replacing aspartic acid with asparagine at position 114 restored reactivity with all five previously non-reactive monoclonal antibodies.
- Substitutions at positions 26 and 47 did not affect antibody binding.
- Human IgE binding was not significantly altered by the amino acid change at position 114.
Conclusions:
- Monoclonal antibodies can distinguish between Der p 2 isoforms, enabling studies on natural isoform occurrence.
- For reliable allergen measurement assays, monoclonal antibodies that do not discriminate between isoforms should be selected.