Transferring Luminex® cytokine assays to a wall-less plate technology: Validation and comparison study with plasma and cell culture supernatants

Versteeg, Leroy; Guezennec, Xavier Le; Zhan, Bin; Liu, Zhuyun; Angagaw, Minilik; Woodhouse, Janice D; Biswas, Subhabrata; Beaumier, Coreen M.

doi:10.1016/j.jim.2016.11.003

Cited by 26 publications

(23 citation statements)

References 20 publications

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“…To measure IFN-␥ and IL-4 in the supernatants, a sandwich enzyme-linked immunosorbent assay (ELISA) method was employed, using mouse IFN-␥ and IL-4 ELISA kits (eBioscience) per the manufacturer's instructions. To measure secreted levels of IL-2, IL-10, IL-12, and TNF-␣, a Luminex-based assay was used, as previously described, that utilizes a Luminex kit from Bio-Rad and a wall-less 96-well plate from Curiox (68). Cytokine concentrations in the supernatant were calculated based on a standard curve, and for each sample duplicate wells were averaged.…”

Section: Methodsmentioning

confidence: 99%

Vaccine-Linked Chemotherapy Improves Benznidazole Efficacy for Acute Chagas Disease

et al. 2018

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Chagas disease affects 6 to 7 million people worldwide, resulting in significant disease burdens and health care costs in countries of endemicity. Chemotherapeutic treatment is restricted to two parasiticidal drugs, benznidazole and nifurtimox. Both drugs are highly effective during acute disease but are only minimally effective during chronic disease and fraught with significant adverse clinical effects. In experimental models, vaccines can be used to induce parasite-specific balanced T1/T2 immune responses that effectively reduce parasite burdens and associated inflammation while minimizing adverse effects. The objective of this study was to determine the feasibility of vaccine-linked chemotherapy for reducing the amount of benznidazole required to significantly reduce blood and tissue parasite burdens. In this study, we were able to achieve a 4-fold reduction in the amount of benznidazole required to significantly reduce blood and tissue parasite burdens by combining the low-dose benznidazole with a recombinant vaccine candidate, Tc24 C4, formulated with a synthetic Toll-like 4 receptor agonist, E6020, in a squalene oil-in-water emulsion. Additionally, vaccination induced a robust parasite-specific balanced T1/T2 immune response. We concluded that vaccine-linked chemotherapy is a feasible option for advancement to clinical use for improving the tolerability and efficacy of benznidazole.

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Section: Methodsmentioning

confidence: 99%

Vaccine-Linked Chemotherapy Improves Benznidazole Efficacy for Acute Chagas Disease

et al. 2018

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“…BAL fluid and lung tissue supernatants were thawed, and cytokine levels were quantitated by using a Bio-Rad (Hercules, CA) Pro mouse cytokine T H 1/T H 2 (IFN-␥, IL-4, IL-5, IL-10, IL-13, IL-17, and TNF-␣) kit with 96-well DropArray DA-bead plates and a DropArray LT210 washing station MX instrument from Curiox (San Carlos, CA), as previously described (59). The results were acquired on a Luminex Magpix instrument using Bio-Plex MP and Bio-Plex Manager software.…”

Section: Methodsmentioning

confidence: 99%

Ascaris Larval Infection and Lung Invasion Directly Induce Severe Allergic Airway Disease in Mice

et al. 2018

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(roundworm) is the most common helminth infection globally and a cause of lifelong morbidity that may include allergic airway disease, an asthma phenotype. We hypothesize that larval migration through the lungs leads to persistent airway hyperresponsiveness (AHR) and type 2 inflammatory lung pathology despite resolution of infection that resembles allergic airway disease. Mice were infected with by oral gavage. Lung AHR was measured by plethysmography and histopathology with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stains, and cytokine concentrations were measured by using Luminex Magpix. -infected mice were compared to controls or mice with allergic airway disease induced by ovalbumin (OVA) sensitization and challenge (OVA/OVA).-infected mice developed profound AHR starting at day 8 postinfection (p.i.), peaking at day 12 p.i. and persisting through day 21 p.i., despite resolution of infection, which was significantly increased compared to controls and OVA/OVA mice. -infected mice had a robust type 2 cytokine response in both the bronchoalveolar lavage (BAL) fluid and lung tissue, similar to that of the OVA/OVA mice, including interleukin-4 (IL-4) ( < 0.01 and < 0.01, respectively), IL-5 ( < 0.001 and < 0.001), and IL-13 ( < 0.001 and < 0.01), compared to controls. By histopathology, -infected mice demonstrated early airway remodeling similar to, but more profound than, that in OVA/OVA mice. We found that larval migration causes significant pulmonary damage, including AHR and type 2 inflammatory lung pathology that resembles an extreme form of allergic airway disease. Our findings indicate that ascariasis may be an important cause of allergic airway disease in regions of endemicity.

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“…The supernatant samples were tested for levels of IL-2, IL-4, IL-5, IL-10, IL-12(p70), GM-CSF, IFN-γ and TNF-α using a Bio-Plex Pro Mouse Th1/Th2 8-plex kit (Bio-Rad, Hercules). To save material and costs, and to increase the sensitivity of the experiment, the kit was used in combination with DA-Bead plates (Curiox Biosystems, Singapore) as previously described [37]. Samples were run on a Bio-Plex Magpix multiplex reader according to manufacturer's recommendations (Luminex, Austin).…”

Section: Methodsmentioning

confidence: 99%

Yeast-expressed recombinant As16 protects mice against Ascaris suum infection through induction of a Th2-skewed immune response

et al. 2017

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BackgroundAscariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB) as an adjuvant, but the exact protective mechanism was not well understood.Methodology/Principal findingsAs16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14) in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99.6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36.7%) and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000) and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel), known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein’s inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response.Conclusions/SignificanceYeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum egg challenge that associates with a Th2-skewed immune response, suggesting that rAS16 could be a feasible vaccine candidate against ascariasis.

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Transferring Luminex® cytokine assays to a wall-less plate technology: Validation and comparison study with plasma and cell culture supernatants

Cited by 26 publications

References 20 publications

Vaccine-Linked Chemotherapy Improves Benznidazole Efficacy for Acute Chagas Disease

Vaccine-Linked Chemotherapy Improves Benznidazole Efficacy for Acute Chagas Disease

Ascaris Larval Infection and Lung Invasion Directly Induce Severe Allergic Airway Disease in Mice

Yeast-expressed recombinant As16 protects mice against Ascaris suum infection through induction of a Th2-skewed immune response

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