Effect of the Protein Corona on Antibody–Antigen Binding in Nanoparticle Sandwich Immunoassays

Abstract

We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, KDeff. Results show that corona formation mediates the specificity and sensitivity of the antibody–antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

Challenges of the Nano–Bio Interface in Lateral Flow and Dipstick Immunoassays

“…Recently, the impact of the protein corona around NP-Ab in paper-based immunoassays was examined. [48] Anecdotally it is known that running strips in buffer solution often does not work because false test lines occur; this effect can be prevented by running strips in human serum. The NP-Ab in the serum forms a protein corona, and the corona is partially responsible for preventing non-specific adsorption to the test line, reducing false positives.…”

“…For example, Langmuir isotherms modified to include surface terms can be used to separate the impact of the surface modifications on antibody-antigen binding in planar and NP surfaces, and can also be applied to LFAs. [48, 52, 53]…”

Frizzled7 Antibody‐Functionalized Nanoshells Enable Multivalent Binding for Wnt Signaling Inhibition in Triple Negative Breast Cancer Cells

“…[42] Using this model, we found an effective dissociation constant of 4.83E-10 M for FZD7-NS and 1.38E-8 M for free FZD7 antibodies (Figure 3B). The lower effective dissociation constant for FZD7-NS compared to free FZD7 antibodies indicates that FZD7-NS have approximately a 100-fold increased binding affinity to FZD7 cell surface receptors relative to freely delivered FZD7 antibodies.…”

“…Data was fit to a modified Langmuir isotherm model as described by Puig et al . [42] to find the effective dissociation constant using the equation $\text{OD}=\frac{{\text{Ab}}_{\text{conc}}}{{\mathrm{K}}_{\mathrm{D}}^{\text{eff}}+{\text{Ab}}_{\text{conc}}}$. In this equation, Ab conc is the concentration of antibody added to cells and ${\mathrm{normalK}}_{\mathrm{normalD}}^{\text{eff}}$ is the effective dissociation constant.…”

Repurposing Old Antibodies for New Diseases by Exploiting Cross Reactivity and Multicolored Nanoparticles

“…with immobilized pDENV and pZIKV exhibited Langmuir-like curves, increasing with NS1 and then saturating. 34 Fits to a modified Langmuir isotherm (red and blue lines, Figure 3b-g) 35 allowed calculation of the LODs, which ranged from 1.8 -972.4 ng/mL ( Table 1). The YFV LOD was below reported values for positive samples (178-4600 ng/mL), supporting its utility in diagnostic assays.…”