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We demonstrate a simple dual-mode multiplexed array-in-well immunoassay for simultaneous classification and detection of serum IgG and IgM antibodies against influenza A and human adenoviruses based on the color and position of the upconversion luminescence on the array. Biotinylated influenza A/H1N1 and A/H5N1 as well as adenovirus serotype 2 and 5 hexon antigens along with positive and negative controls were printed in an array format onto the bottom of streptavidin-coated microtiter wells. The anti-influenza A and antiadenovirus antibodies present in the sample were captured to the array and detected with antihuman antibody-coated upconverting nanophosphors (UCNPs). The green emitting UCNPs (NaYF4:Yb(3+),Er(3+)) coated with antihuman IgG and blue emitting UCNPs (NaYF4:Yb(3+),Tm(3+)) coated with antihuman IgM were used to detect human IgG and IgM antibodies, respectively. The emission of the bound UCNPs was imaged free of autofluorescence with anti-Stokes photoluminescence microwell imager. No spectral cross-talk was observed between green and blue emitting UCNPs. Also the cross-reactivities between UCNP-conjugates and immobilized human IgG and IgM antibodies were negligible. Position of the signal on the array defined the antigen specificity and the antibody class was defined by the color of the upconversion luminescence. This technology could be used for differentiation between acute infection from past infection and immunity. Additionally, the class of the antibody response can be used for the differentiation between primary and secondary infections, hence, facilitating epidemiological seroprevalence studies.

Upconverting nanoparticles (UCNPs) are attractive reporters in immunoassays because of their outstanding detectability. However, non-specific binding of antibody-UCNP conjugates on protein coated solid support results in background, which limits the immunoassay sensitivity. Thus, the full potential of UCNPs as reporters cannot be fully exploited. The authors report here a method to improve the sensitivity of UCNP-based immunoassays by reducing the non-specific binding of antibody-UNCP conjugates on the protein coated solid support. In the assays studied here, poly(acrylic acid) (PAA) coated NaYF:Yb,Er type UCNPs were conjugated to two different antibodies against cardiac troponin I (cTnI) and thyroid stimulating hormone (TSH). The two-step heterogeneous sandwich immunoassays were performed in microtitration wells, and the green luminescence of antibody-UCNP conjugates was measured at 540 nm upon 980 nm excitation. Non-specific binding of antibody-UCNP conjugates was reduced by mixing free PAA with PAA coated UCNPs before adding the UCNPs to the wells. The free PAA in the buffer reduced the background in both cTnI and TSH immunoassays (compared to the control assay without free PAA). The limits of detection decreased from 2.1 ng·L to 0.48 ng·L in case of cTnI and from 0.070 mIU·L to 0.020 mIU·L in case of TSH if PAA is added to the buffer. Presumably, the effect of free PAA is due to blocking of the surface areas where PAA coated UCNP would bind proteins non-specifically. The method introduced here is likely to be applicable to other kinds of PAA-coated nanoparticles, and similar approaches conceivably work also with other nanoparticle coatings. Graphical abstract The presence of free poly(acrylic acid) (PAA) in a buffer solution prevents aggregation and non-specific protein binding of PAA-coated upconverting nanoparticles (UCNPs) in heterogeneous sandwich immunoassays. The decrease in non-specific binding enables distinctly more sensitive assays to be performed.

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