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.
Cancer antigen 125 (CA125) is a widely used biomarker in monitoring of epithelial ovarian cancer (EOC). Due to insufficient cancer specificity of CA125, its diagnostic use is severely compromised. Abnormal glycosylation of CA125 is a unique feature of ovarian cancer cells and could improve differential diagnosis of the disease. Here we describe the development of a quantitative lateral flow immunoassay (LFIA) of aberrantly glycosylated CA125 which is widely superior to the conventional CA125 immunoassay (CA125IA). With a 30 min read-out time, the LFIA showed 72% sensitivity, at 98% specificity using diagnostically challenging samples with marginally elevated CA125 (35–200 U/mL), in comparison to 16% sensitivity with the CA125IA. We envision the clinical use of the developed LFIA to be based on the substantially enhanced disease specificity against the many benign conditions confounding the diagnostic evaluation and against other cancers.
Detection of hepatitis B Virus surface antigen (HBsAg) is an established method for diagnosing both acute and chronic hepatitis B virus (HBV) infection. In addition to enzyme immunoassays (EIAs), rapid diagnostic tests (RDTs) are available for the detection of HBsAg in resource-poor settings. However, the available RDTs have inadequate sensitivity and therefore are not suitable for diagnosis of patients with low levels of HBsAg and for blood screening. To provide a high-sensitivity RDT, we developed a lateral flow immunoassay (LFIA) for HBsAg utilizing upconverting nanoparticle (UCNP) reporter. The UCNP-LFIA can use whole blood, serum, or plasma and the results can be read in 30 min using a reader device. When compared with a commercial conventional visually read LFIA, the developed UCNP-LFIA had a Limit of Detection (LoD) of 0.1 IU HBsAg/ml in spiked serum, whereas the LoD of the conventional LFIA was 3.2 IU HBsAg/ml. The developed UCNP-LFIA fulfills the WHO criterion for blood screening (LoD ≤ 0.13 IU HBsAg/ml) in terms of LoD. The UCNP-LFIA and conventional LFIA were evaluated with well-characterized sample panels. The UCNP-LFIA detected 20/24 HBsAg-positive samples within the HBsAg Performance Panel and 8/10 samples within the Mixed Titer Performance Panel, whereas the conventional LFIA detected 8/24 and 4/10 samples in these panels, respectively. The performance of the assays was further evaluated with HBsAg-positive (n = 108) and HBsAg-negative (n = 315) patient samples. In comparison with a central laboratory test, UCNP-LFIA showed 95.4% (95% CI: 89.5–98.5%) sensitivity whereas sensitivity of the conventional LFIA was 87.7% (95%CI: 79.9–93.3%).
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