Standard curves for each of the 5 analytes.

<p>The standard curves generated after 5-PL regression analysis across seven twofold dilutions of the Liquichek 3 standards for each assay in the multiplexed array. AGP, CRP, RBP and sTfR assay plots are from the basic antigen competitor assays and show concomitant decrease in chemiluminescence as the concentration of analyte is increased reflecting the non-labeled antigen out-competing the biotinylated antigen. In the ferritin sandwich assay, the chemiluminescence increases with analyte concentration reflecting the increased binding of the labeled secondary antibody to the captured ferritin. AGP, alpha-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor.</p

Bland-Altman analysis.

<p>Bland-Altman plots of differences between log concentrations (y-axes) versus the average concentrations (x-axes) for each analyte in 72 plasma specimens assayed using the Quansys MMAT and conventional assays. Center solid line indicates mean difference; upper and lower solid lines indicate the 95% confidence interval. AGP, alpha-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor.</p

Assay precision, ranges, lower limits of detection (LOD) and assay linearity for MMAT.

<p>AGP, alpha-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor; ID, iron deficiency; VAD, vitamin A deficiency; MMAT, multiplexed micronutrient assessment tool; CV, coefficient of variation.</p><p>Assay precision, ranges, lower limits of detection (LOD) and assay linearity for MMAT.</p

Standard concentrations.

<p>The revised concentration of the Liquichek 3 control analytes in the test well as measured via the conventional ELISAs (mean ± SD). The standard spans points 1–7 of subsequent twofold dilutions with 8, a diluent only negative control, completing the set. AGP, alpha-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor.</p><p>* The mean concentrations provided with the Liquichek 3 product insert.</p><p>Standard concentrations.</p

Effects of sample type and addition of EDTA to diluent.

<p>AGP, alpha-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor.</p

Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings

Assay precision and limits of detection (LOD) for conventional ELISAs.

<p>Coefficients of variation (CV) for the conventional ELISA kits against which the multiplexed micronutrient assessment tool (MMAT) was compared. AGP, alpha-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor.</p>†<p>For RBP, precision given as average intra- and inter-assay CVs in the calibrated range.</p><p>*For sTfR, 3 values are presented from the 6 reported for intra- and 12 provided for inter-assay CV in the kit insert.</p><p>Assay precision and limits of detection (LOD) for conventional ELISAs.</p

Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings

Fig. 1A. The arrangement of multiplexed assays in each well.

<p>A, alpha-1-acid glycoprotein; C, C-reactive protein; F, ferritin; R, retinol binding protein 4; sT, soluble transferrin receptor. <b>Fig. 1B. An image of 8 array wells demonstrating the chemiluminescence generated from assay spots.</b> The top left hand well (well 1) indicates the highest concentration of standard followed by six subsequent two-fold dilutions (wells 2 through 7). The final lower right hand well (well 8) is the buffer-only negative control.</p

DNA amplification by Recombinase Polymerase Amplification.

<p>The three core proteins, recombinase, single-strand DNA binding protein (SSB) and strand-displacing polymerase enable PCR-like DNA amplification without the need for thermal cycling or an initial chemical or thermal melting step. This diagram was created by TwistDx Ltd (<a href="http://www.twistdx.co.uk/our_technology/" target="_blank">http://www.twistdx.co.uk/our_technology/</a>) and is licensed under a Creative Commons Attribution 3.0 United States License.</p