A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

Kinetic titration method in flow cytometry for quantification of cell receptors.

For the quantitative determination of cell receptors by fluorescence flow cytometry, we proposed a new method, which takes into account the reaction kinetics. The binding reaction of the ligand with receptors begins after placing the cells in the ligand solution. In the proposed method, there are several samples with the same concentration of cells and different initial concentrations of fluorescently labeled ligand, and each sample is measured by a flow cytometer once at the time when the following condition is met: the product of the incubation time (cells with ligand) and the initial concentration of ligand is the same for all samples. The proposed approach eliminates disadvantages and combines advantages of both kinetic and titration methods for quantification of receptors on single cells without the use of traditional calibration fluorescent beads. Practical application of the method was demonstrated in quantification of CD8 and CD14 on peripheral blood human leukocytes. Particularly, we found decreased (by a factor of two) mean number of CD14 on monocytes and granulocytes in patients with atherosclerosis (treated in the hospital) compared to conditionally healthy donors, whereas no difference was found in the mean CD8 expression on leukocytes between the same patient and donor groups.

Self-assembly strategy to reduce non-specific adsorption for the development of high sensitivity quantitative immunoassay.

The development of functionalized surfaces with low non-specific adsorption is important for their biomedical applications. To inhibit non-specific adsorption on glass substrate, we designed a novel optical biochip by modifying a layer of dense negatively charged film (SO32-) on its substrate surface via self-assembly. Compared with the untreated glass substrate, it reduced the adsorption by about 300-fold or 400-fold by poly (styrene sulfonic acid) sodium salt (PSS), or meso-tetra (4-sulfonatophenyl) porphine dihydrochloride (TSPP) on individually the modified glass substrate. Considering the effect of fluorescence resonance energy transfer (FRET) between TSPP and the QDs in solution by mixing, a strategy of 2-layer of TSPP followed by 4-layer of PSS was designed to modify the glass for preparing biochips. Under the optimized conditions, the biochip on functionalized glass substrate co-treated with TSPP and PSS realized the sensitive quantitative detection of C-reactive protein (CRP) based on a quantum dot fluorescence immunosorbent assay (QD-FLISA). The limit of detection (LOD) for CRP achieved 0.69 ng/mL with the range of 1-1,000 ng/mL using TSPP and PSS co-treated glass substrate surface, which was respectively about 1.9-fold and 7.5-fold more sensitive to the PSS-modified biochip and the TSPP-modified biochip. This work demonstrated an effective and convenient strategy to obtain biochips with low non-specific adsorption properties on functionalized surfaces, thus providing a new approach for creating ultra-high sensitivity microchannels or microarrays on glass substrates.

A new testing platform using fingerstick blood for quantitative antibody response evaluation after SARS-CoV-2 vaccination.

Testing and vaccination have been major components of the strategy for combating the ongoing COVID-19 pandemic. In this study, we have developed a quantitative anti-SARS-CoV-2 spike (S1) IgG antibody assay using a fingerstick dried blood sample. We evaluated the feasibility of using this high-throughput and quantitative anti-SARS-CoV-2 spike (S1) IgG antibody testing assay in vaccinated individuals. Fingerstick blood samples were collected and analyzed from 137 volunteers before and after receiving the Moderna or Pfizer mRNA vaccine. Anti-SARS-CoV-2 S1 IgG antibody could not be detected within the first 7 days after receiving the first vaccine dose, however, the assay reliably detected antibodies from day 14 onwards. In addition, no anti-SARS-CoV-2 nucleocapsid (N) protein IgG antibody was detected in any of the vaccinated or healthy participants, indicating that the anti-SARS-CoV-2 S1 IgG assay is specific for the mRNA vaccine-induced antibodies. The S1 IgG levels detected in fingerstick samples correlated with the levels found in venous blood plasma samples and with the efficacy of venous blood plasma samples in the plaque reduction neutralization test (PRNT). The assay displayed a limit of quantification (LOQ) of 0.59 µg/mL and was found to be linear in the range of 0.51-1000 µg/mL. Finally, its clinical performance displayed a Positive Percent Agreement (PPA) of 100% (95% CI: 0.89-1.00) and a Negative Percent Agreement (NPA) of 100% (95% CI: 0.93-1.00). In summary, the assay described here represents a sensitive, precise, accurate, and simple method for the quantitative detection and monitoring of post-vaccination anti-SARS-CoV-2 spike IgG responses.

Cytokine Levels in Human Vitreous in Proliferative Diabetic Retinopathy.

In this study, we compare the vitreous cytokine profile in patients with proliferative diabetic retinopathy (PDR) to that of patients without PDR. The identification of novel cytokines involved in the pathogenesis of PDR provides candidate therapeutic targets that may stand alone or work synergistically with current therapies in the management of diabetic retinopathy. Undiluted vitreous humor specimens were collected from 74 patients undergoing vitrectomy for various vitreoretinal disorders. Quantitative immunoassay was performed for a panel of 36 neuroinflammatory cytokines in each specimen and assessed to identify differences between PDR (n = 35) and non-PDR (n = 39) patients. Levels of interleukin-8 (IL-8), IL-15, IL-16, vascular endothelial growth factor (VEGF), VEGF-D, c-reactive protein (CRP), serum amyloid-A (SAA), and intracellular adhesion molecule-1 (ICAM1) were significantly increased in the vitreous of PDR patients compared to non-PDR patients (p < 0.05). We report novel increases in IL-15 and IL-16, in addition to the expected VEGF, in the human vitreous humor of patients with PDR. Additionally, we confirm the elevation of ICAM-1, VCAM-1, SAA, IL-8 and CRP in the vitreous of patients with PDR, which has previously been described.

Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms.

Purpose: Lacritin is a tear glycoprotein with pro-tearing and pro-ocular surface homeostasis activities that is selectively deficient in most dry eye tears. Proteoforms include an active monomer, inactive polymers, and a splice variant termed lacritin-c. Quantitation of the different proteoforms of tear lacritin may provide a diagnostic tool for ocular diseases. Here, we report the development of an immunoassay for the quantification of multiple lacritin proteoforms in human tear samples. Methods: Basal tears collected on Schirmer test strips with anesthesia were eluted by diffusion and centrifugation under optimized conditions. Tear protein concentrations were determined, and 2.56 µg of each sample was separated by SDS-PAGE followed by western blot analysis. Blots were challenged with anti-Pep Lac N-term antibodies. Detection was with fluorescent secondary antibodies visualized by the LI-COR Odyssey CLx imaging system and quantified with standard curves of recombinant lacritin. Results: The percent total lacritin (ng lacritin/100 ng total protein) ranged from 1.8% to 14.8%. Monomer, lacritin-c, and polymer proteoform percent total protein ranged from 1.1% to 6.3%, 0.3% to 5.4%, and 0.7% to 5.7%, respectively. Monomer lacritin was detected at concentrations of 6 to 176 µM, with lacritin-c and polymer proteoforms at 2 to 46 µM and 1 to 23 µM, respectively. Conclusions: This assay greatly exceeds the power and sensitivity of our prior lacritin enzyme-linked immunosorbent assay that was not capable of distinguishing monomer from polymers and lacritin-c proteoforms. Translational Relevance: A new method has been developed to quantitate multiple proteoforms of tear lacritin in preparation for analyses of samples from clinical trials.

Blocking-free and self-contained immunoassay platform for one-step point-of-care testing.

This paper reports a quantitative and sensitive one-step point-of-care testing (POCT) chip built on a perfluorinated substrate patterned with polydopamine microspots array. The capture antibody was covalently immobilized on the polydopamine microspots, while the fluorescently labelled detection antibody was physically adsorbed on the perfluorinated surface. The POCT chip allowed one-step sandwich immunoassay and was able to directly detect the analytes from the whole blood without sample preprocessing. By further taking advantages of the strong fluorescence quenching ability of the polydopamine, the blocking-free substrate was able to achieve similar performance in detecting and quantifying the protein biomarkers as the substrate with the blocking treatment. The blocking-free strategy not only made the fabrication of the chip simple and convenient, but also improved the chip's sensitivity for biomarker quantification. Finally, we demonstrated that the self-contained POCT platform maintained the performance for one-step immunoassay even after long-term storage. With the POCT platform, we are one step closer to a sample-in-answer-out diagnostic system.

LEGENDplex™: Bead-assisted multiplex cytokine profiling by flow cytometry.

Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunology-based therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.

Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes.

We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand-receptor (antigen-antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct ( k+= (5.6 ± 0.2) × 107 M-1 min-1 ) and reverse ( k-= (1.3 ± 0.2) × 10-2 min-1 ) rate constants of ligand-receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

Portable GMR Handheld Platform for the Detection of Influenza A Virus.

Influenza A virus (IAV) is a common respiratory pathogen infecting many hosts including humans, pigs (swine influenza virus or SIV), and birds (avian influenza virus or AIV). Monitoring swine and avian influenza viruses in the wild, farms, and live poultry markets is of great significance for human and veterinary public health. A portable, sensitive, and quantitative immunoassay device will be of high demand especially in the rural and resource-limited areas. We report herein our Z-Lab point-of-care (POC) device for sensitive and specific detection of swine influenza viruses with minimum sample handling and laboratory skill requirements. In the present study, a portable and quantitative immunoassay platform based on giant magnetoresistive (GMR) technology is used for the detection of IAV nucleoprotein (NP) and purified H3N2v. Z-Lab displays quantitative results in less than 10 min with sensitivities down to 15 ng/mL and 125 TCID50/mL for IAV nucleoprotein and purified H3N2v, respectively. This platform allows lab-testing to be performed outdoors and opens up the applications of immunoassays in nonclinical settings.

Synthesis of Reabsorption-Suppressed Type-II/Type-I ZnSe/CdS/ZnS Core/Shell Quantum Dots and Their Application for Immunosorbent Assay.

We report a phosphine-free one-pot method to synthesize ZnSe/CdS/ZnS core-shell quantum dots (QDs) with composite type-II/type-I structures and consequent reabsorption suppression properties. The as-synthesized QDs possess high efficient red emission (with quantum yield of 82%) and high optical stability. Compared to type-I QDs, the ZnSe/CdS/ZnS QDs show larger Stokes shift and lower reabsorption which can reduce the emission loss and improve the level of fluorescence output. The ZnSe/CdS/ZnS QDs are used as fluorescent labels to exploit their application in fluorescence-linked immunosorbent assay (FLISA) for the first time in the detection of C-reactive protein (CRP) with a limit of detection (LOD) of 0.85 ng/mL, which is more sensitive than that of CdSe/ZnS type-I QDs based FLISA (1.00 ng/mL). The results indicate that the ZnSe/CdS/ZnS type-II/type-I QDs may be good candidates for applications in biomedical information detection.

Comparing Assay Performance of ELISA and Chemiluminescence Immunoassay in Detecting Antibodies to Hepatitis B Surface Antigen.

INTRODUCTION: Antibodies to Hepatitis B surface Antigen (Anti-HBs) levels are measured as markers for immune response to vaccination and in decision making for post-exposure prophylaxis against Hepatitis-B. Several immunoassay formats are used to measure Anti-HBs, thus carrying the possibility of variation in measured levels between different assays. This study compares the performance of Chemiluminescence Immunoassay (CLIA) against Enzyme-linked Immunosorbent Assay (ELISA) in measuring Anti-HBs titer by looking into concordance between the two test reports. AIM: To compare the agreement between ELISA and CLIA in measurement of Anti-HBs antibody titers. MATERIALS AND METHODS: This prospective comparative study conducted at Kasturba Medical College, Manipal measured consecutive serum samples (69) sent for anti-HBs levels during May-June 2016 using both CLIA (Abbott Architect) and ELISA (Bio-Rad). Anti-HBs values of ≤10mIU/ml was considered as non-protective and >10mIU/ml as protective. The agreement between the tests in classifying the antibody titers as non-protective or protective was computed using Kappa coefficient, and the difference in individual titer values between the tests compared using Bland-Altman plot on SPSS (v.15). RESULTS: Out of the 69 samples analysed, 18 samples (26.1%) were of health-care personnel and remaining of patients. Agreement between ELISA and CLIA in identifying the antibody titers as protective and non-protective were 96.5% and 90.9% respectively, resulting in an agreement of 0.84. The coefficient-of-variation of ELISA and CLIA were 74.5% and 113.1%, respectively. Three value based discordant results were noted; two samples deemed protective by ELISA were reported as non-protective by CLIA. One non-protective titer by ELISA was reported as protective by CLIA. CONCLUSION: Analytical agreement is good between the two immunoassays. However there are some discrepancies in quantitative measurement. This may have been due the variation in the standard calibrators used in each assay. Though CLIA showed more variation in the values, it has the advantage of being automated test with low turn around time. Therefore, both the test methodologies can be reliably used in place of each other for detection of Anti- HBs titer.

A homogeneous immunosensor for AFB1 detection based on FRET between different-sized quantum dots.

Mycotoxins are fatal threats in food safety due to their strong carcinogenesis and toxicity, thus requiring highly sensitive detections. Herein, different-sized quantum dots (QDs) were used to construct a Förster resonance energy transfer (FRET) based immunosensor for sensitive detection of aflatoxin B1 (AFB1) in rice grains. To avoid irregular aggregation between two kinds of QDs, monovalent monoclonal antibody (mAb)-labeled red QDs (~0.84 anti-AFB1 mAbs per QD) and multivalent hapten-labeled green QDs (~6.8 AFB1 per QD) were designed as acceptor and donor, respectively. The anti-AFB1 mAbs and AFB1 interactions promoted one or more acceptors bound with a multivalent AFB1-labeled donor, resulting in energy transfer from the green QDs to the red QDs. Various parameters that influence the immunoassay including reactant ratio of donor to acceptor, buffer pH value, buffer ionic strength and immunoreaction time were systematically investigated and optimized. With optimal conditions, the obtained energy transfer efficiency is proportional to the logarithm of AFB1 concentration in a range over 0.19-16 pM (0.06-5 ng/mL), while offering a limit of detection of 0.13 pM (0.04 ng/mL) in rice extracts. The recovery rates of the intra-assay for spiked samples at AFB1 concentrations of 0.1, 1.0, and 5.0 ng/mL were 83.27% ± 3.27%, 97.36% ± 4.55% and 83.04% ± 4.94%, respectively, and those for the inter-assay were 81.28% ± 6.11%, 95.97% ± 7.07%, and 82.78% ± 5.99%, respectively. Statistical analysis using t-test had no significant difference between the proposed FRET-based immunoassay and the commercial enzyme-linked immunosorbent assay kit.