A Quantitative Holographic Agglutination Assay for Immunoglobulin A

TL;DR

The paper tackles the need for rapid, label-free quantification of IgA in clinical samples by introducing a Holographic Agglutination Assay (HAA) that uses Jacalin to induce selective IgA agglutination. It leverages Total Holographic Characterization (THC) in inline holographic video microscopy to measure per-particle diameter $d_p$, refractive index $n_p$, and agglutinate counts, enabling quantitative IgA readouts in a physiologically relevant, 10× diluted range. Key findings show a linear relationship between IgA concentration and agglutinate number density within the detectable range, clear IgA selectivity over IgG and non-glycosylated controls, and informative morphology data that act as a fingerprint for lectin–glycoprotein interactions. The work proposes a general, bead-free platform for lectin-mediated holographic agglutination with potential to monitor other immunoglobulins or glycoproteins in clinical samples, reducing reagent complexity and enabling broader diagnostic applications.

Abstract

This study introduces a Holographic Agglutination Assay for quantifying levels of the immunoglobulin protein IgA in biological samples. This is the first example of a label-free and bead-free assay that quantifies protein agglutinates by direct detection using Total Holographic Characterization. A proof-of-concept assay for human serum immunoglobulins is demonstrated using Jacalin, the galactose-specific plant lectin, to induce selective agglutination. By analyzing the size, refractive index, and number of particles in an assay sample, we obtain a reproducible and quantitative measurement of galactosylated immunoglobulins in a given sample. The assay is calibrated for a physiologically relevant reference interval of IgA concentrations in a 10x diluted emulated biological sample from low (80 mg/dL, 5 μM) to high (320 mg/dL, 20 μM) levels. The assay clearly distinguishes samples containing IgA from samples containing IgG. More broadly, this study introduces a platform for creating lectin-mediated Holographic Agglutination Assays to monitor levels of immunoglobulins in biological samples. The ability to quantify immunoglobulin levels efficiently in clinical samples is likely to be valuable for diagnostics and will provide a basis for assaying other proteins that can be induced to agglutinate.

Figures (4)

• Figure 1: Schematic of the Holographic Agglutination Assay. The agglutination assay is initiated by incubating the tetrameric protein, lectin, with the glycoprotein immunoglobulin A (upper left) in a microfuge tube. Gal represents the O-linked galactose on IgA that binds to the lectin Jacalin to form protein agglutinates. The microfluidic cell shown (lower left) can be used to quantify the Human serum IgA levels in each sample well. The optical setup (middle) used for Total Holographic Characterization to produce and record holograms of the agglutinates as they flow through the microfluidic channel. Representative distribution plot (right) of all detected particles in a single measurement depicting the values of diameter and refractive index from the fitted holograms.
• Figure 2: Proof-of-concept demonstration of an agglutination assay for human IgA. Scatter plots show the diameter ($d_p$) and refractive index ($n_p$) of each particle in a sample. (a) Background: Jacalin (1 in PBS buffer, 2 sample). (b) Background: human IgA (0.5 in PBS buffer, 2 sample). (c) Assay: Jacalin (1) incubated with human IgA (0.2) for 30 in PBS buffer, 0.75 sample). Each data point represents a single detected particle. Particle count increases more than 10-fold due to agglutination of lectin and IgA. White circles denote mean values of the distributions.
• Figure 3: Quantitative holographic agglutination assay for human serum IgA in the presence of Jacalin. Each point in the scatter plot represents the number density of detected particles as a function of ligand concentration for a fixed concentration of the lectin Jacalin,0.5. Human IgA (circles) forms agglutinates in the presence of Jacalin and the number density of agglutinates increases linearly with IgA concentration over the clinically relevant range. Human IgG (squares) does not increase the number density of detected particles above background due to the lack of terminal galactose. Similarly, alcohol dehydrogenase (triangles) does not generate a signal above background and also serves as a negative control.
• Figure 4: Diameter and refractive index of Jacalin agglutinates by particle morphology. Spherical, rod-like and other-shaped agglutinates formed after incubation of Jacalin with human serum IgA (a) and IgG (b). Negative control with alcohol dehydrogenase from yeast (c). All experiments with immunoglobulins were conducted in triplicates. The size of the marker represents the average particle count.