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

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, <i>K</i>_D^eff. 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

Surface-Enhanced Raman Spectroscopy-Based Sandwich Immunoassays for Multiplexed Detection of Zika and Dengue Viral Biomarkers

Zika and dengue are mosquito-borne diseases that present similar nonspecific symptoms but possess dramatically different outcomes. The first line of defense in epidemic outbreaks are rapid point-of-care diagnostics. Because many outbreaks occur in areas that are resource poor, assays that are easy to use, inexpensive, and require no power have become invaluable in patient treatment, quarantining, and surveillance. Paper-based sandwich immunoassays such as lateral flow assays (LFAs) are attractive as point-of-care solutions as they have the potential for wider deployability than lab-based assays such as PCR. However, their low sensitivity imposes limitations on their ability to detect low biomarker levels and early diagnosis. Here, we exploit the high sensitivity of surface-enhanced Raman spectroscopy (SERS) in a multiplexed assay that can distinguish between Zika and dengue nonstructural protein 1 (NS1) biomarkers. SERS-encoded gold nanostars were conjugated to specific antibodies for both diseases and used in a dipstick immunoassay, which exhibited 15-fold and 7-fold lower detection limits for Zika NS1 and dengue NS1, respectively. This platform combines the simplicity of a LFA with the high sensitivity of SERS and could not only improve Zika diagnosis but also detect diseases sooner after infection when biomarker levels are low

Phylogenetic analysis of DENV-2 based on the complete genome sequence.

<p>The evolutionary history was inferred using the Maximum Likelihood method, using the General Time Reversible model for nucleotide substitution with discrete Gamma distribution to model evolutionary rate differences among sites [4 categories (+<i>G</i>, parameter = 0.45)]. The tree with the highest log likelihood (−46330.60) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 91 nucleotide sequences with a total of 10,167 positions in the final dataset. A total of 1000 bootstrap replicates were run and values ≥99 are represented as percentage in respective nodes. The Brazilian DENV-2 lineages are shown in grey. For clarity purposes some branches were collapsed. VE/CO/GU/BZ/MX 1999–2009 contains isolates from Venezuela (VE61095/2007, DENV-2/VE/BID-V2944/2005, -V2424/2004, -V2492/200, -V2216/2003, -V2262/2006), Colombia (DENV-2/CO/BID-V3370/2004, -V3369/1999, -V1603/2004), Guatemala (FDA-GUA09/2009), Belize (BZ/BID-V2952/2002) and Mexico (DENV-2MX/BID-V3661, -V3714 and –V3768); PR 1986–1995 contains isolates from Puerto Rico (DENV-2/US/BID-V1356/1993, -V855/1992, -V1182/1989, -V1175/1988, -V1183/1990, -V1171/1987, -V1164/1986, DENV-2/PR/17DN/1995 and DENV-2/PR/6780DN/1994 ) and PR 1994–2007 also contains isolates from Puerto Rico (DENV-2/US/BID-V37DN/1994, -V1424/1996, -V1427/1999, -V1398/1997, -V1038/1998, -V1367/1995, -V1463/2000, V1472/2001, -V593/2005 and –V1412/2007). Evolutionary analyses were conducted in MEGA5.0.</p

Bayesian coalescent and discrete phylogeography analyses of Brazilian DENV-2 based on envelope nucleotide sequence.

<p>Subtree of the maximum clade credibility tree was inferred using 144 DENV-2 envelope sequences (1,485 nt). The subtree containing isolates of the American/Asian genotype is displayed here. Time of the most recent common ancestor (MRCA) was estimated using the year of isolation as the calibration point, under the relaxed molecular clock, with the Tamura Nei Model, with discrete Gamma distribution and an estimated nucleotide substitution rate of 7.5⁻⁴. The posterior probabilities values ≥0.96 are represented by (*) and values ≥0.99 are represented by (**), inside the nodes. The years that the MRCA was estimated to exist are shown for some nodes with upper and lower intervals in parenthesis. The origin value of the reverse scale axis corresponds to year 2010. Using different colors, (legend shown on the left side), terminal branches were annotated based on geographic location of DENV-2 isolates. Internal nodes of the tree which presented modal state posterior probability ≥0.60 were also colored according to their most probable location states, inferred by discrete phylogeographical analysis. Brazilian lineages are delimited by square brackets. For clarity purposes some branches were collapsed. SJRP/2008 contains 11 isolates from São José do Rio Preto/São Paulo/Brazil (DENV-2/BR/BID-V3653/2008, -V3638/2008, -V3640/2008, -V3483/2008, -V3637/2008, -V3495/2008, -V3645/2008, -V3481/2008, -V3486/2008, -V3650/2008 and -V3648/2008); CU/US/KN 1997–2001 contains isolates from Cuba (Cuba115/97 and Cuba165/1997), Puerto Rico (US/BIC-V1387/1998) and Saint Kitts and Nevis (KN/BID-V2951/2001); VE/CO/GU/BZ/MX 1999–2009; PR 1986–1995 and PR 1994–2007 contain the same isolates as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059422#pone-0059422-g001" target="_blank">Figure 1</a> caption. Analyses were performed using programs from BEAST package v.1.6.1, BEAUTi, Tracer v.1.5.0, TreeAnotator v.1.6.1 and FigTree v.1.3.1 (B) Map of Brazil showing the macro-regions and states. The black pin indicates the approximate location of São José do Rio Preto/São Paulo/Brazil.</p

Amino acid differences in the envelope protein of Brazilian DENV-2 lineages.

1<p>- except HQ012515.BR59382/RN/1997 and HQ012518.BR66985/RJ/2000 (K).</p>2<p>- except DENV-2/BR/BID-V2386/2003, DENV-2/BR/BID-V2396/2006, JF804028.BR/DB015/2006 (V).</p

Rank of immunogenicity of DENV-3 sequences.

<p>Thirty-three DENV-3 sequences were submitted to B and T epitope prediction using bioinformatics servers and further classified according to their immunogenic potential. Sequences previously clustered in B and C clades have epitopes, in envelope, NS1, NS2a and NS5, putatively less immunogenic when compared to the same epitopes of sequences clustered in clade A, except for sequence ACY70777.1. Substitutions in the capsid were irrelevant.</p

A comparison of nanoparticle-antibody conjugation strategies in sandwich immunoassays

<p>Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.</p

Genetic analysis per genomic region.

*<p>Total number of mutations.</p>**<p>Nucleotide diversity.</p>***<p>Average number of nucleotide differences.</p

Non-neutral codons according to the assayed methods (SLAC and REL) at the specified significance levels (p = 0.05 and Bayes factor = 50, respectively).

<p>Non-neutral codons according to the assayed methods (SLAC and REL) at the specified significance levels (p = 0.05 and Bayes factor =  50, respectively).</p

Positively-selected codon transformations tracked over a clade credibility (MMC) tree.

<p>Light gray boxes represent SJRP clades. Numbers indicates codon position and one-letter symbols indicate amino-acids. Transformations for each character can be evaluated as follows: • Unique transformation all over the tree; ▪ character state fixed along that branch; ◂ same transformation occurs in another branch (homoplasy); ▸ transformation(s) in the same character occurs along that branch; ⧫ same transformation occurs in another branch (homoplasy) and transformation(s) in the same character occur along that branch.</p