Published online Dec 21, 2018. doi: 10.3748/wjg.v24.i47.5379
Peer-review started: July 13, 2018
First decision: August 27, 2018
Revised: September 1, 2018
Accepted: October 5, 2018
Article in press: October 5, 2018
Published online: December 21, 2018
Celiac disease is a chronic immune mediated disorder of the small intestine caused by consuming the protein gluten present in wheat and some other cereals. Following the activation of the innate immune system, a number of cytokines as well as antibodies are released in celiac patients that can be used as specific biomarkers to develop diagnostic tests. Over the years, a number of diagnostic tests have been developed, however, in spite of the good initial results in terms of sensitivity and specificity, when these tests are used on a large scale they have lowered predictive values. In the present study, a novel assay using peptide functionalised gold nanoparticles was developed that can be useful in an exclusion based diagnostic strategy.
The number of celiac disease sufferers are rapidly increasing throughout the world, and there is an increased need for newer detection methods that are easy to use, accurate as well as cheaper to enable early identification of the disease. The present study addresses this issue through the development of a novel assay combining the unique properties of gold nanoparticles with the specificity of the antibodies. The developed assay shows great potential to be developed as a point-of-care test that would be beneficial for large scale screening of celiac disease.
In order to develop a celiac diagnostic assay based on the properties of the gold nanoparticles combined with the specificity of the antibodies from serum the following aims have been addressed in this work: (1) To develop method for the binding and adsorption of peptide derived from gliadin, the main antigenic protein causing celiac disease on the surface of gold nanoparticles; (2) to detect and measure the concentrations of antibodies to be used as biomarkers in serum; (3) to test and validate the developed test on real patient serum samples.
The peptide coated gold nanoparticles were characterized using UV-vis spectra, dynamic light scattering and transmission electron microscopy. The UV-vis absorbance readings of peptide coated AuNPs following interactions with AGA and IgG from rabbit serum (control antibody) were used to calculate the percentage absorbance values. The students t-test was used to compare the sets of quantitative data that were collected independently of one another to calculate the p value and determine statistical significance. The assay sensitivity was determined based on the colorimetric response values.
The clinical accuracy of the peptide coated AuNPs was determined using a selected, varied set of thirty human serum samples obtained from patients with celiac disease or controls without celiac disease. The results for the thirty clinical samples were recorded after the visual examination of precipitate formation and the determination of a shift or change in absorbance values using a UV-vis spectrophotometer. The assay sensitivity was determined based on the colorimetric response values obtained for each serum sample.
The peptide sequence was successfully coated to the AuNP and characterization methods indicated that a stable colloidal suspension of the peptide coated AuNPs was achieved that was sustained by the high affinity biotin-avidin interactions.
The addition of anti-gliadin antibody to peptide coated AuNPs as well as in spiked serum at a threshold level resulted in lowering as well as a shift of absorbance peaks that indicated the aggregation of AuNPs. On the other hand, in the presence of a non-specific, normal IgG antibody there was no interaction between the peptide coated AuNPs, with no reduction in color or shift in wavelength or aggregation of AuNPs.
The clinical accuracy of the peptide coated AuNPs was tested using 30 clinical samples, where 26 samples were shown to have the same analysis as that obtained with existing serology and histology, however, 2 false positive results and 2 false negative results were also obtained using the AuNP-peptide-AGA assay giving the AuNP-peptide-AGA assay an overall accuracy of 86.6%.
In this study, we demonstrate the potential of peptide functionalised gold nanoparticles as a colorimetric sensor for screening celiac disease. The AuNP-peptide assay seems promising for development as a point-of-care test, this is because it is based on the formation of a precipitate as well as a reduction in color for a positive sample in the presence of a celiac disease specific autoantibody, thereby, eliminating the need for secondary antibody. This greatly reduces the cost of development for the assay and would be a step in the direction of one-step detection for celiac disease based on single antibody detection.
The AuNP-Peptide based approach shows great potential and would be particularly useful in aiding large-scale screening of the general population, particularly in the pre-selection of young Celiac disease (CD) sufferers which can be then confirmed by mucosal biopsy. A positive result would strengthen the possibility of CD while a negative test would help avoid unnecessary intestinal biopsy thereby reducing the economic burden on healthcare resources resulting in cost savings.