Leukotriene C4/D4/E4 ELISA Kit (DEIA-XYL1)

Regulatory status: For research use only, not for use in diagnostic procedures.

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Size
96T
Sample
biological fluid
Intended Use
This is an ELISA (Enzyme-Linked ImmunoSorbent Assay) for the quantitative analysis of Leukotriene C4/D4/E4 levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and Leukotriene C4/D4/E4 in the sample for a limited number of binding sites.
Contents of Kit
1. EIA BUFFER: 30 mL. Provided to dilute enzyme conjugate and Leukotriene C4 standards. NOTE: PLEASE ONLY USE THE EIA BUFFER THAT IS PROVIDED WITH THIS KIT LOT.
2. WASH BUFFER (10X): 20 mL. Dilute 10-fold with deionized water. Diluted wash buffer is used to wash all unbound enzyme conjugate, samples and standards from the plate after the one hour incubation.
3. K-BLUE SUBSTRATE: 20 mL. Stabilized 3,3', 5,5' Tetramethylbenzidine (TMB) plus Hydrogen Peroxide (H2O2) in a single bottle. It is used to develop the color in the wells after they have been washed. Keep refrigerated. LIGHT SENSITIVE.
4. EXTRACTION BUFFER (5X): 30mL. Dilute 5-fold with deionized water. This is used for diluting extracted and non-extracted samples.
5. LEUKOTRIENE C4/D4/E4 ENZYME LYOPHILIZED CONJUGATE: Two vials of lyophilized LTC4/D4/E4 horseradish peroxidase conjugate. Reconstitution with 75 µL of deionized water results in a 50:1 concentrate. Store lyophilized vials at -20°C. Store reconstituted vials at 4°C. Blue capped vials.
6. LEUKOTRIENE C4 STANDARD: 100 µL. Leukotriene C4 standard at the concentration of 1 µg/mL. Green capped vial.
7. LEUKOTRIENE C4/D4/E4 ANTIBODY-COATED MICROPLATE: A 96 well Maxisorp™ Nunc microplate with anti-LTC4/D4/E4 monoclonal rat antibody precoated on each well. The plate is ready to use as is. DO NOT WASH!
Storage
Lyophilized conjugate: -20°C or less
Do not freeze kit reconstituted conjugate
All other kit components: 4°C
General Description
Leukotriene C4/D4/E4(LTC4/D4/E4) are potent mediators of immediate hypersensitivity, bronchoconstriction, smooth muscle contraction, increased vascular permeability and epithelial mucous secretion. They are derived from arachidonic acid through the 5-lipoxygenase pathway. The monoclonal antisera in this kit recognizes LTC4, LTD4 and LTE4 for a combined quantitative value.
Standard Curve

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References


Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

PLOS ONE

Authors: Kumar, Ramakrishnan B.; Purhonen, Pasi; Hebert, Hans; Jegerschold, Caroline

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LOin vitro.

The Overlooked Storm in Coronavirus Disease 2019 (COVID-19)?

AMERICAN JOURNAL OF PATHOLOGY

Authors: Hammock, Bruce D.; Wang, Weicang; Gilligan, Molly M.; Panigrahy, Dipak

Severe coronavirus disease 2019 (COVID-19) symptoms, including systemic inflammatory response and multisystem organ failure, are now affecting thousands of infected patients and causing widespread mortality. Coronavirus infection causes tissue damage, which triggers the endoplasmic reticulum stress response and subsequent eicosanoid and cytokine storms. Although proinflammatory eicosanoids, including prostaglandins, thromboxanes, and leukotrienes, are critical mediators of physiological processes, such as inflammation, fever, allergy, and pain, their roles in COVID-19 are poorly characterized. Arachidonic acid-derived epoxyeicosatrienoic acids could alleviate the systemic hyperinflammatory response in COVID-19 infection by modulating endoplasmic reticulum stress and stimulating the resolution of inflammation. Soluble epoxide hydrolase (sEH) inhibitors, which increase endogenous epoxyeicosatrienoic acid levels, exhibit potent anti-inflammatory activity and inhibit various pathologic processes in preclinical disease models, including pulmonary fibrosis, thrombosis, and acute respiratory distress syndrome. Therefore, targeting eicosanoids and sEH could be a novel therapeutic approach in combating COVID-19. In this review, we discuss the predominant role of eicosanoids in regulating the inflammatory cascade and propose the potential application of sEH inhibitors in alleviating COVID-19 symptoms. The host-protective action of omega-3 fatty acid-derived epoxyeicosanoids and specialized proresolving mediators in regulating anti-inflammation and antiviral response is also discussed. Future studies determining the eicosanoid profile in COVID-19 patients or predinical models are pivotal in providing novel insights into coronavirus-host interaction and inflammation modulation.

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