Dermatophagoides pteronyssinus 1 (Der p1) is a major mite allergen in Dermatophagoides pteronyssinus house dust mite. Currently there are no reliable ready-to-use-kits for the determination of Der p1. Therefore we developed a ready-to-use-ELISA for Der p1.
A ninety-six-wells plate (NUNC Maxisorp) was coated with a monoclonal antibody (clone 9-5-1) against Der p1 generated in a mouse. The monoclonal-antibody was obtained from a hybrid between BALB/c spleen cells and a myeloma cell line. Samples and a standard are incubated for one hour at room temperature. For the standard we used a semi-purified standard obtained from Der p1 whole culture material. Next, a rabbit-polyclonal antibody (from rabbits immunized with Der p1) was used and incubated for 30 minutes, followed by a peroxidase-labeled detection antibody and substrate for detection. Afterwards cleavage of the substrate was quantified by measuring the optical density at 450 nm using a microplate reader. A pre-coated plate and all other components are included in the kit.
The working range of the Der p1 ELISA was between 1 ng/ml and 15 ng/ml. The Lowest Limit of Quantification (LLOQ) was 1 ng/ml. The CV’s of the accuracy and precision were lower than 15%. The ELISA kit was also tested on robustness, specificity, sensitivity, repeatability and stability. All of these variables were tested according the EMEA guidelines for validation. The ELISA kit shows a good result for each of these variables. There is a small cross-reactivity (5%) against E maynei.
This ELISA kit is useful for analyzing unknown samples for Dermatophagoides pteronyssinus mite allergens and shows 5% cross-reactivity only with Euroglyphus maynei due to genetic similarity. There is no other cross-reactivity with any other mite species for example Der f1, Der p2, or Acarus siro.
