Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E.
Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>>>>>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.
Division of Nutritional Sciences, School of Biosciences, University of Nottingham, Sutton Bonington campus, Loughborough, LE12 5RD, UK.
N K
NK
Renault
S R
SR
Gaddipati
F
F
Wulfert
F H
FH
Falcone
L
L
Mirotti
P J
PJ
Tighe
V
V
Wright
M J C
MJ
Alcocer
D000485:Allergens / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D002457:Cell Extracts; D019295:Computational Biology; D001330:Electronic Data Processing; D005512:Food Hypersensitivity / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D057166:High-Throughput Screening Assays; D006801:Humans; D007136:Immunoglobulins / Q000097:blood*; D008962:Models, Theoretical; D011237:Predictive Value of Tests; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D012680:Sensitivity and Specificity; D006113:United Kingdom
D000485:Allergens; D002457:Cell Extracts; D007136:Immunoglobulins; D011506:Proteins