IgG and IgM differentiation in a particle-based agglutination assay by control over antigen surface density

Abstract

Point-of-care (POC) testing offers fast and on-site diagnostics and can be crucial against many infectious diseases and in screening. One remaining challenge in serological POC testing is the quantification of immunoglobulin G (IgG) and immunoglobulin M (IgM). Quantification of IgG/IgM can be important to evaluate immunity and to discriminate recent infections from past infections and primary infections from secondary infections. POC tests such as lateral flow immunoassays allow IgG and IgM differentiation; however, a remaining limitation is their incapacity to provide quantitative results. In this work, we show how samples containing IgG or IgM can be distinguished in a nanoparticle-based agglutination biosensing assay by tuning the density of antigens on the nanoparticles' surface. We employ direct STochastic Optical Reconstruction Microscopy to quantify the accessible SARS-CoV-2 trimeric spike proteins conjugated to magnetic nanoparticles at a single-particle level and gain insight into the protein distribution provided by the conjugation procedure. Furthermore, we measure the anti-SARS-CoV-2 IgG/IgM induced agglutination using an optomagnetic readout principle. We show that particles with high antigen density have a relatively higher sensitivity toward IgM compared to IgG, whereas low antigen density provides a relatively higher sensitivity to IgG. The finding paves the way for its implementation for other agglutination-based serology tests, allowing for more accurate disease diagnosis.

FIG. 1.

Assessing the structure–function relationship for protein-conjugated MNPs. (a) We aim to establish if varying the density of protein per MNP can influence the detection of antibodies. (b) MNPs were conjugated using TCO-TZ click chemistry on BSA-passivated MNPs. (c) Conjugated nanoparticles were characterized for accessible protein using dSTORM to determine the density of active sites. Top image: diffraction limited TIRF image. Bottom image: reconstructed dSTORM image (scale bar 100 nm). (d) The conjugates were further tested with an immunomagnetic assay on a commercial BluSense instrument. MNPs functionalized with COVID antigen are allowed to react with the COVID antibody present in the biological media. Agglutination of MNP causes antibodies to bind to MNPs under the influence of a magnetic field, thus forming chains. MNP chains repeatedly align and relax in an oscillating magnetic field modulating the transmittance of light measured using the lock-in technique (optomagnetic signal).

FIG. 2.

Characterization of MNP conjugates using dSTORM super-resolution microscopy. (a) MNP conjugates were immobilized on a BSA/BSA-biotin coated glass coverslip. (b) Images of D150, D300, D700, and D1400 conjugates in TIRF (left) and reconstructed dSTORM images showing densities of localizations (scale bar is 1000 nm). (c) The number of localizations per particle recorded during the length of the dSTORM movie. (d) The number of bound antibodies calculated using calibration. Dots represent individual MNP conjugates.

FIG. 3.

Performances of MNPs in an optomagnetic assay. (a) Dose–response of high (D150) and low (D700) conjugates with mAb IgG in PBST. (b) Dose–response curve with a dilution series of patient sample high in IgG in buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween, and pH 8). (c) Dose–response curve with a dilution series of a patient sample infected with COVID-19 containing IgM. (d) D150/D700 ratio of the OM signal obtained with high-(D150) and low-density conjugate (D700) for Mouse IgG, high IgG, IgM samples, and patient samples. The x-axis shows the OM signal for D700.

FIG. 4.

Performance of MNPs against samples taken from COVID-19-infected patients. (a) and (b) The OM signal obtained with D150 and D700 conjugates for two patient sample series collected post-infection. The patient samples were diluted 20× in SDB. (c) The D150/D700 ratio for patient 1 and 2 samples, plotted against the time after infection. The patient 1 sample lacks data points for day 56 because only data points with D150 values above limit of detection are shown.

FIG. 5.

Mechanistic interpretation of the structure–function relationship. IgG and IgM cross-link MNPs differently for different binding site densities. (a) High-density MNPs (D150) at low IgG concentration mainly bind to one MNP, which gradually reduces when IgG concentration increases. (b) Low-density MNPs (D700) for low IgG concentration have free Fab domains that can cross-link to other MNPs, which further increases at high IgG concentration until all proteins on D700 are occupied. (c) and (d) At low IgM concentrations, the high-density conjugate (D150) forms larger aggregates due to the availability of more Fab-binding domains. The low-density conjugate (D700) cross-links to IgM molecules at low concentrations with smaller aggregates due to the availability of fewer binding sites compared to D150. At higher IgM concentrations, the OM signal increases and saturates for both conjugates.