Allergome microarray for detection of total repertoire af allergen-specific IgE in human serum

Abstract

Allergy, or type 1 hypersensitivity, is defined as a malfunction of immune system where a person’s body is hypersensitized to react immunologically to non-immunogenic substances. These substances are usually innocuous environmental antigens such as pollen, house dust mites, animal epithelia, moulds, etc and collectively are known as allergens. Type I hypersensitivity is characterised by excessive activation of mast cells by IgE resulting in a systemic inflammatory response. Therefore, elevated levels of allergen specific IgEs in the serum is the key in the pathogenesis of allergic diseases. The worldwide prevalence of allergy is dramatically increasing and it is of particular concern in industrialized countries. Fifty million Americans suffer from allergic diseases including asthma, rhinitis, sinusitis, dermatitis and food allergy and in the UK it affects one in four people at some point in their lives. Therefore, there is an increasing need for a sensitive in vitro test capable of rapidly and accurately quantify allergen-specific IgE from patient sera. The aim of this project is to assess the potential of high throughput technology for simultaneous in vitro measurement of allergen-specific IgEs to a variety of well-known environmental and food allergens and the development of a novel diagnostic biochip for clinical use. For this purpose, a protein microarray platform based on non purified allergens has been developed, evaluated, and validated to generate proof-of- principle and to demonstrate the specificity and sensitivity of the array design. The protein microarray method developed for detection of specific IgE antibodies in human sera consist of four defined steps: 1. Printing: microspots containing total allergen extract and, antibodies are covalently immobilized onto activated glass microscope slides by means of high speed robotics. Minute volumes reagents are arrayed in 7 X 7 matrices, each spot diameter being 400 pm. 2. Processing: slides are initially developed with small volumes (100 pl_) of serum samples. A seguential series of reagents are applied to the slide including detecting antibody and amplification reagents conjugated to a fluourophore. The total assay time required is less than 3 hours. 3. Scanning slides are processed by means of a high sensitivity fluorescence-detecting scanner that is able to gather the fluorescence signal emitted by each array component. 4. Quantification and analysis: computational analysis of the image data. The studies carried out to validate the assay have shown that by using the tyramide amplification system it is possible to achieve an excellent degree of analytical sensitivity and analytical specificity. In addition, no cross-reactivity to other immunoglobulins at normal serum levels was detected. Reproducibility and repeatability are also within the standard requirements for this kind of immunoassay. Short-term stability studies have been performed to assess the stability of the assay over-time and the results are satisfactory over 90 days. Finally, the clinical validation of the microarray assay showed that the data generated with this system is substantially better than the reference method in terms of sensitivity, correlation between class score and fluorescent signal. The microarray technology has several advantages over existing diagnostic methods such as ELISA. This system offers high throughput and true parallelism; it is highly economical in the use of specimens and reagents and also provides a rapid and accurate detection method for the quantification of specific IgEs in human serum. The knowledge and experience gained in this project will be used in the evaluation of a pre-market microarray- based in vitro diagnostic device.