SciCombinator

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Concept: Immunoassay

149

A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = -0.4287x + 0.3132 (R² = 0.9904). The working range was 0.07-2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%-111.9% and 91.7%-114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R² = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.

Concepts: Nanoparticle, Nanotechnology, ELISA, ELISPOT, Assay, Immunoassay, Magnetic nanoparticles, Magnetic immunoassay

141

The diagnostic work-up for heparin induced thrombocytopenia (HIT) can take several days. Consequently patients may be speculatively switched onto replacement anticoagulant therapy before a diagnosis is confirmed. On-demand immunoassay diagnostic testing enables timely treatment decisions, based on test results.

Concepts: Medical terms, Diagnosis, Anticoagulant, Heparin, Thrombocytopenia, Immunoassay, Medical test, Heparin-induced thrombocytopenia

58

Background To estimate the frequency and duration of detectable Zika virus (ZIKV) RNA in human body fluids, we prospectively assessed a cohort of newly infected participants in Puerto Rico. Methods We evaluated samples obtained from 150 participants (including 55 men) in whom ZIKV RNA was detected on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay in urine or blood in an enhanced arboviral clinical surveillance site. We collected serum, urine, saliva, semen, and vaginal secretions weekly for the first month and then at 2, 4, and 6 months. All specimens were tested by means of RT-PCR, and serum was tested with the use of anti-ZIKV IgM enzyme-linked immunosorbent assay. Among the participants with ZIKV RNA in any specimen at week 4, biweekly collection continued until all specimens tested negative. We used parametric Weibull regression models to estimate the time until the loss of ZIKV RNA detection in each body fluid and reported the findings in medians and 95th percentiles. Results The medians and 95th percentiles for the time until the loss of ZIKV RNA detection were 14 days (95% confidence interval [CI], 11 to 17) and 54 days (95% CI, 43 to 64), respectively, in serum; 8 days (95% CI, 6 to 10) and 39 days (95% CI, 31 to 47) in urine; and 34 days (95% CI, 28 to 41) and 81 days (95% CI, 64 to 98) in semen. Few participants had detectable ZIKV RNA in saliva or vaginal secretions. Conclusions The prolonged time until ZIKV RNA clearance in serum in this study may have implications for the diagnosis and prevention of ZIKV infection. Current sexual-prevention guidelines recommend that men use condoms or abstain from sex for 6 months after ZIKV exposure; in 95% of the men in this study, ZIKV RNA was cleared from semen after about 3 months. (Funded by the Centers for Disease Control and Prevention.).

Concepts: Sexual intercourse, ELISA, ELISPOT, Assay, Eva Engvall, Immunoassay, Multiplex, Body fluids

51

This work demonstrates that a full laboratory-quality immunoassay can be run on a smartphone accessory. This low-cost dongle replicates all mechanical, optical, and electronic functions of a laboratory-based enzyme-linked immunosorbent assay (ELISA) without requiring any stored energy; all necessary power is drawn from a smartphone. Rwandan health care workers used the dongle to test whole blood obtained via fingerprick from 96 patients enrolling into care at prevention of mother-to-child transmission clinics or voluntary counseling and testing centers. The dongle performed a triplexed immunoassay not currently available in a single test format: HIV antibody, treponemal-specific antibody for syphilis, and nontreponemal antibody for active syphilis infection. In a blinded experiment, health care workers obtained diagnostic results in 15 min from our triplex test that rivaled the gold standard of laboratory-based HIV ELISA and rapid plasma reagin (a screening test for syphilis), with sensitivity of 92 to 100% and specificity of 79 to 100%, consistent with needs of current clinical algorithms. Patient preference for the dongle was 97% compared to laboratory-based tests, with most pointing to the convenience of obtaining quick results with a single fingerprick. This work suggests that coupling microfluidics with recent advances in consumer electronics can make certain laboratory-based diagnostics accessible to almost any population with access to smartphones.

Concepts: Antibody, Infectious disease, Type I and type II errors, ELISA, ELISPOT, Assay, HIV test, Immunoassay

46

Early detection of HIV infection is the best way to prevent spread of the disease and to improve the efficiency of the antiretroviral therapy. Nucleic acid amplification tests (NAAT) have become the gold-standard for detecting low-concentrations of the virus in blood. However, these methods are technically demanding and cost-prohibitive in developing countries. Immunoassays are more affordable and can be more easily adapted for point-of-care diagnosis. However, the sensitivity so far of these methods has been too low. We here report the development of a sandwich immunoassay that combines nanomechanical and optoplasmonic transduction methods for detecting the HIV-1 capsid antigen p24 in human serum. The immunoreactions take place on the surface of a compliant microcantilever where gold nanoparticles are used as both mechanical and plasmonic labels. The microcantilever acts as both a mechanical resonator and an optical cavity for the transduction of the mechanical and plasmonic signals. The limit of detection of the immunoassay is 10-17 g/mL that is equivalent to one virion in 10 mL of plasma. This is 5 orders of magnitude better than last generation of approved immunoassays and 2 orders of magnitude better than NAAT. This technology meets the demands to be produced en masse at low cost and the capability for miniaturization to be used at the point-of-care.

Concepts: Antiretroviral drug, HIV, AIDS, Immune system, Viral load, Infectious disease, Gold, Immunoassay

34

In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10(-18) g ml(-1). p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.

Concepts: Antibody, ELISA, ELISPOT, Assay, Gold, HIV test, Immunoassay, Secondary antibody

28

Male fertility problems range from diminished production of sperm, or oligozoospermia, to nonmeasurable levels of sperm in semen, or azoospermia, which is diagnosed in nearly 2% of men in the general population. Testicular biopsy is the only definitive diagnostic method to distinguish between obstructive (OA) and nonobstructive (NOA) azoospermia and to identify the NOA subtypes of hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome. We measured by selected reaction monitoring assay 18 biomarker candidates in 119 seminal plasma samples from men with normal spermatogenesis and azoospermia, and identified two proteins, epididymis-expressed ECM1 and testis-expressed TEX101, which differentiated OA and NOA with high specificities and sensitivities. The performance of ECM1 was confirmed by enzyme-linked immunosorbent assay. On the basis of a cutoff level of 2.3 μg/ml derived from the current data, we could distinguish OA from normal spermatogenesis with 100% specificity, and OA from NOA with 73% specificity, at 100% sensitivity. Immunohistochemistry and an immunoenrichment mass spectrometry-based assay revealed the differential expression of TEX101 in distinct NOA subtypes. TEX101 semen concentrations differentiated Sertoli cell-only syndrome from the other categories of NOA. As a result, we propose a simple two-biomarker decision tree for the differential diagnosis of OA and NOA and, in addition, for the differentiation of NOA subtypes. Clinical assays for ECM1 and TEX101 have the potential to replace most of the diagnostic testicular biopsies and facilitate the prediction of outcome of sperm retrieval procedures, thus increasing the reliability and success of assisted reproduction techniques.

Concepts: Sperm, ELISA, ELISPOT, Assay, Spermatozoon, Semen, Immunoassay, Multiplex

28

Enzyme-linked immunosorbent assays (ELISA) are being used extensively for the identification of Bt-protein in Bt transgenic crops. A sandwich ELISA test is the most preferable immunoassay for the quantification of Bt-protein in transgenic cotton plants. Here, we describe development of sandwich ELISA, employing polyclonal rabbit antibody as a capture antibody and HRP-labeled mouse anti-Bt protein-antibody as a detector antibody.

Concepts: Antibody, ELISA, ELISPOT, Assay, Immunoassay, Multiplex, Magnetic immunoassay

28

This work describes the quick and simple fabrication of toner-based 96-microzone plates by a direct-printing technology. The printer deposits a toner layer (ca. 5 μm thick) on the polyester surface which acts as a hydrophobic barrier to confine small volumes of sample on test zones (wells). A 96-microzone toner plate was explored to demonstrate its capability of performing enzyme-linked immunosorbent assay (ELISA). The detection of anti-immunoglobulin G (anti-IgG) and immunoglobulin M (IgM) antibodies has been successfully achieved in cell culture and serum samples, respectively. The use of a conventional microplate reader has allowed obtaining a limit of detection of 13 fmol of mouse IgG per zone on printed microplates. The IgM antibody has been detected in a serum sample collected from a patient infected with dengue virus. The detection of a primary infection has been provided by a microplate reader and also by a cell phone camera. Besides the bioanalytical feasibility, toner-based zones have shown good repeatability for inter-zone and intra-plate comparisons. The relative standard deviation (RSD) values for inter-zone (n = 12) and intra-plate (n = 3) comparisons were lower than 6% and 11%, respectively. Furthermore, it was found that the lifetime of each printed microplate depends on the storage temperature. The shelf life for devices stored at 10 °C has been estimated to be ca. four weeks.

Concepts: Immune system, Antibody, Immunology, ELISA, ELISPOT, Assay, Immunoassay, Microtiter plate

28

A novel SERS-based sandwich immunoassay using DNA aptamers, silica-encapsulated hollow gold nanospheres (SEHGNs) and a gold-patterned microarray was developed for sensitive detection of VEGF (vascular endothelial growth factor) angiogenesis protein markers. Here, a DNA aptamer conjugated to SEHGN was used as a highly reproducible SERS-encoding nanoprobe, and a hybrid microarray including hydrophilic gold wells and other hydrophobic areas was used as a SERS substrate. Target specific DNA aptamers that fold into a G-quadruplex structure were used as a target recognition unit instead of VEGF antibodies. The detection sensitivity was increased by 2 or 3 orders of magnitude over the conventional ELISA method. In particular, the dynamic concentration range was 3 or 4 orders of magnitude greater than that of conventional ELISA. The results demonstrate that this sensing strategy using DNA aptamers is a powerful platform for the design of novel immune-sensors with high performance. In particular, SERS-based detection using SEHGNs provides great promise for highly sensitive biomarker sensing with unprecedented advantages.

Concepts: Antibody, DNA, Protein, Angiogenesis, Vascular endothelial growth factor, ELISA, Immunoassay, Aptamer