The main objective of this study was to analyze and compare recombinant timothy grass allergen diagnostic performance on microarray chip. Timothy grass allergen components Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or in N. benthamiana. In total 113 patient sera samples were used to assess allergy microarray chip inter-assay variability, diagnostic sensitivity and specificity, and correlation, for the detection of allergen-specific IgE levels. The majority of recombinant timothy grass allergens showed high or very high positive correlation of immunoreactivity with other commercial multiplex tests. Maltose binding protein fused Phl p 11 showed high specificity and sensitivity, with 0.3 arbitrary cut-off value. High intraassay variation reflects allergen-dependent degradation. Additionally, a sIgE prevalence to timothy grass allergens was assessed within a tested patient cohort and showed a similar sensitization profile among multiple studies.

Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. MAbs were proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed. The allergen was detected and its concentration was determined in only 3 of 6 analyzed HDM allergen extracts from different manufacturers. HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy.