Fabrication of Microarrays for the Analysis of Serological Antibody Isotypes against Food Antigens

Abstract

Food intolerance is delayed adverse food reactions which follow consumption of specific foods. The underlying mechanisms are not well understood, but food intolerance is often considered as a type 2 hypersensitivity reaction mediated by immunoglobulin G (IgG) antibody. To understand the causes of food intolerance, it is important to investigate sensitization patterns of food-specific IgGs (sIgG) in relation to dietary patterns and physical conditions. Conventional approaches to measure serological IgGs often require large volumes of serum, thus are not suitable for highly multiplexed assays. To overcome this impracticality, we developed a highly sensitive method to screen the sIgGs and other antibody isotypes against 66 antigens with minimal amount of serums. We prepared a microarray by immobilizing food antigens on activated glass slides. Human sera and their dietary information were obtained from 30 subjects. Aliquots (200 nl) of sera were analyzed against 66 food antigens in parallel. sIgG levels were determined and analyzed in relation to subjects’ dietary patterns. The levels of antibody isotypes were also examined to understand the relationship between allergy and food intolerance. The developed microarray showed exceptional performances in antibody screening and demonstrated the potential to be used as an automated assay system.

AHR rs4410790 genotype and IgG levels: Effect modification by lifestyle factors

“…Serum IgG levels were measured by arrays of protein G and A at different concentrations [ 21 , 22 ]. Immunoassays were performed using the microarrays as follows; each chamber was treated with 2% BSA in PBST for 1 h, washed with PBST, and incubated with 200 nL of diluted human serum (1/400) in PBST for 1 h. The microarray was then washed with PBST and treated sequentially with a solution of detection antibody and a solution of Cy3-conjugated goat anti-human IgG in PBST for 1 h. The fluorescence intensity was measured using a GenePix 4000B (Axon Instruments, Union city, CA) using an excitation wavelength of 532 nm and an emission wavelength of 570 nm.…”