Method Description

Immunoglobulin A:

Total IgA levels are measured by immunonephelometry. In this Siemens Nephelometer II method, the light scattered onto the antigen-antibody complexes is measured. The intensity of the measured scattered light is proportional to the amount of antigen-antibody complexes in the sample under certain conditions. If the antibody volume is kept constant, the signal behaves proportionally to the antigen volume. A reference curve is generated by a standard with a known antigen content on which the scattered light signals of the samples can be evaluated and calculated as an antigen concentration. Antigen-antibody complexes are formed when a sample containing antigen and the corresponding antiserum are put into a cuvette. A light beam is generated with a light emitting diode, which is transmitted through the cuvette. The light is scattered onto the immuno-complexes that are present. Antigen and antibody are mixed in the initial measurement, but no complex is formed yet. An antigen-antibody complex is formed in the final measurement. The result is calculated by subtracting value of the final measurement from the initial measurement. The distribution of intensity of the scattered light depends on the ratio of the particle size of the antigen-antibody complexes to the radiated wavelength.(Instruction manual: Siemens Nephelometer II, Siemens, Inc.; Version 3, 2008; Addendum to the Instruction Manual 2.3, 08/2017)

Tissue Transglutaminase IgA/IgG Antibodies:

IgA and IgG antibodies to tissue transglutaminase (tTG) are detected by enzyme-linked immunosorbent assay (ELISA). Recombinant human tTG antigen expressed in Escherichia coli is adsorbed to wells of a microtiter plate under conditions that preserve the native state of the antigen. Diluted patient sera are added to the microtiter plate wells under conditions that allow binding of the antibodies to the tTG antigen. Unbound sample constituents are washed away, and horseradish peroxidase (HRP)-labeled antihuman IgA or IgG antibody conjugate is added to each well. After a second incubation, unbound HRP-label is removed, and bound conjugate is detected by adding tetramethylbenzidine (TMB) chromogenic substrate. After a final incubation, colored product is measured spectrophotometrically; the absorbance of the patient sample is compared to the positive calibrator. The absorbance is directly proportional to the level of IgA or IgG antibodies to tTG, which is expressed in arbitrary units.(Package inserts: QUANTA Lite R h-tTG IgA and IgG. Inova Diagnostics, Inc; rev. 8, 01/2020)

Deamidated Gliadin IgA/IgG Antibodies:

IgA and IgG antibodies to deamidated gliadin peptides are detected by ELISA. Purified peptides are adsorbed to wells of a microtiter plate under conditions that preserve the native state of the antigen. Diluted patient sera are added to the microtiter plate wells under conditions that allow binding of the antibodies to the deamidated gliadin peptides. Unbound sample constituents are washed away and HRP-labeled antihuman IgA or IgG antibody conjugate is added to each well. After a second incubation, unbound HRP-label is removed, and bound conjugate is detected by adding TMB chromogenic substrate. After a final incubation, colored product is measured spectrophotometrically; the absorbance of the patient sample is compared to the positive calibrator. The absorbance is directly proportional to the level of IgA or IgG antibodies to deamidated gliadin peptides, which is expressed in arbitrary units.(Package inserts: QUANTA Lite Gliadin IgA II; rev.2, 10/2019; QUANTA Lite Gliadin IgG II. INOVA Diagnostics, Inc; Rev.4, 05/2020)

Endomysial IgA Antibody:

Endomysial IgA antibodies are detected by indirect immunofluorescence assay. Frozen sections of rhesus monkey esophagus substrate are overlaid with dilutions of patient sera and incubated. After washing, the slides are overlaid with fluorescein-conjugated IgA antisera and incubated. After a final washing, the slides are analyzed through fluorescence microscopy.(Sheppard AL, Elwenspoek MMC, Scott LJ, et al: Systematic review with meta-analysis: the accuracy of serological tests to support the diagnosis of coeliac disease. Aliment Pharmacol Ther. 2022 Mar;55(5):514-527. doi: 10.1111/apt.16729)