Prostaglandin D2 ELISA Kit

Name: Prostaglandin D2 ELISA Kit
SKU: DEIA5036
Rating: 5 (1 reviews)

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

COA

SDS

Datasheet

Summary

Sample

cell culture lysates, enzymatic reactions

Species Reactivity

Human

Detection Method

cELISA

Intended Use

CD's PGD2 ELISA Kit is a competitive assay that can be used for quantification of PGD2 in cell culture lysates and enzymatic reactions. The assay has a range from 19.5-2,500 pg/ml and a sensitivity (80% B/B0) of approximately 55 pg/ml.

Contents of Kit

1. Prostaglandin D2 EIA Monoclonal Antibody: 1 vial/100 dtn
2. Prostaglandin D2 AChE Tracer: 1 vial/100 dtn
3. Prostaglandin D2 EIA Standard: 1 vial
4. EIA Buffer Concentrate (10×): 2 vials/10 mL
5. Wash Buffer Concentrate (400×): 1 vial/5 mL
6. Polysorbate 20: 1 vial/3 mL
7. Goat Anti-Mouse IgG Coated Plate: 1 plate
8. 96-Well Cover Sheet: 1 cover
9. Ellman's Reagent: 3 vials/100 dtn
10. EIA Tracer Dye: 1 vial
11. EIA Antiserum Dye: 1 vial

Storage

Store at 4°C for frequent use, at -20°C for infrequent use. Avoid multiple freeze-thaw cycles. For more detailed information, please download the following document on our website.

Precision

The intra- and inter-assay CVs have been determined at multiple points on the standard curve.

Intra- and inter-assay variation
* %CV represents the variation in concentration (not absorbance) as determined using a reference standard curve.
+ Outside of the recommended usable range of the assay.

Detection Range

19.5-2,500 pg/ml

Sensitivity

Sensitivity (defined as 80% B/B0) = 55 pg/ml Mid-point (defined as 50% B/B0) = 170-310 pg/ml
The sensitivity and mid-point were derived from the standard curve shown above. The standard was diluted with ELISA Buffer.

General Description

Prostaglandin D2 (PGD2) is biosynthesized in the brain by a soluble, 26 kDa glutathione-independent lipocalin-type PGD2 synthase.This PGD2 accumulates in the cerebrospinal fluid (CSF), where it induces physiologic sleep in rats and humans. PGD2 is also synthesized in mast cells and leukocytes by a cellular, myeloid-type, glutathione-dependent PGD synthase. This PGD2 which is formed in the intracellular and vascular compartments is rapidly metabolized to 11β-PGF2α.Thus, plasma measurements of PGD2 synthesis are most appropriately focused on the measurement of 11β-PGF2α.
Measurement of the parent eicosanoid PGD2 is appropriate in cell culture lysates and in CSF, where concentrations of several hundred pg/ml have been measured.
All studies of PGD2 biosynthesis should take into consideration the chemical instability of PGD2 and its rapid degradation in the presence of serum proteins such as albumin. PGD2 also readily degrades in both acidic and basic media to give a variety of decomposition products including PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2. For these reasons, antibodies against PGD2 have generally been prepared against conjugates of a stable methoximine derivative (i.e., PGD2-MOX). Our PGD2-MOX and PGD2-MOX Express ELISA Kits are based on this chemistry and require the PGD2 in all samples to be converted to the stable methoximine derivative prior to performing the assay. These assays are well suited and recommended for the measurement of PGD2 in complex matrices. The antibody utilized in this PGD2 ELISA was generated in a unique way allowing the direct measure of PGD2 without prior conversion to the methoximine compound.
Biochemistry of Acetylcholinesterase
The electric organ of the electric eel, E. electricus, contains an avid AChE capable of massive catalytic turnover during the generation of its electrochemical discharges. The electric eel AChE has a clover leaf-shaped tertiary structure consisting of a triad of tetramers attached to a collagen-like structural fibril. This stable enzyme is capable of high turnover (64,000 s^-1) for the hydrolysis of acetylthiocholine.
A molecule of the analyte covalently attached to a molecule of AChE serves as the tracer in AChE enzyme immunoassays.
Quantification of the tracer is achieved by measuring its AChE activity with Ellman's Reagent. This reagent consists of acetylthiocholine and 5,5'-dithio-bis-(2-nitrobenzoic acid). Hydrolysis of acetylthiocholine by AChE produces thiocholine. The non-enzymatic reaction of thiocholine with 5,5'-dithio-bis-(2-nitrobenzoic acid) produces 5-thio-2-nitrobenzoic acid, which has a strong absorbance at 412 nm (ε = 13,600).
AChE has several advantages over other enzymes commonly used for enzyme immunoassays. Unlike horseradish peroxidase, AChE does not self-inactivate during turnover. This property of AChE also allows redevelopment of the assay if it is accidentally splashed or spilled. In addition, the enzyme is highly stable under the assay conditions, has a wide pH range (pH 5-10), and is not inhibited by common buffer salts or preservatives. Since AChE is stable during the development step, it is unnecessary to use a 'stop' reagent, and the plate may be read whenever it is convenient.

Standard Curve

Citations

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Background

Prostaglandins are synthesized by arachidonic acid through cyclooxygenase-1, cyclooxygenase-2 and prostaglandin synthase, regulate a variety of physiological systems, and are associated with a variety of diseases. Prostaglandin D2 (PGD2) is synthesized by prostaglandin D synthase (PGDS). The change of hematopoietic prostaglandin D synthase (H-PGDS) may be related to the pathophysiology of asthma. PGD2 can mediate a variety of physiological effects, including inhibition of platelet aggregation and sleep induction. As a powerful bronchoconstrictor, PGD2 has an effect on both asthmatic and non-asthmatic patients and can induce vasodilation. It can also induce mucus secretion in the respiratory tract as an inflammatory mediator. In contrast, PGD2 can have the properties of muscle relaxation and vasoconstriction in other tissues.

Immune cells activated during inflammation, mainly mast cells, release PGD2, while PGD2 interacts with two PGD2 receptors (DP1 and DP2) to stimulate thromboxane receptors at very low concentrations. DP2 is a G protein coupled receptor expressed on the membrane surface of Th2 cells, mast cells and eosinophils. PGD2 and its metabolites can activate DP2 receptors and induce downstream pro-inflammatory signaling pathways, resulting in the activation of Th2 cells and eosinophils and migration to the inflammatory sites of asthma. Activation of DP2 receptors on Th2 cells upregulates the expression of IL-4, IL-5 and IL-13 in a dose-dependent manner, and induces the migration of Th2 cells, while activation of DP2 on eosinophils promotes the migration of these cells and increases the degranulation of eosinophils. IL-4, in synergy with TNF-α, enhances the expression of vascular cell adhesion molecule-1 and P-selectin on vascular endothelial cells and facilitates the trans-endothelial entry of eosinophils from the bloodstream into the respiratory system. IL-5 is involved in the maturation of eosinophils and inhibits apoptosis of these cells.

Activation of DP2 receptor signaling pathway by PGD2 and downstream effects Figure 1. The DP2 receptor pathway and its downstream effects
(Source: Brightling CE, et al. 2020)

Current studies have shown that DP2 is the main regulator in the pathophysiological process of asthma, which can stimulate and amplify the inflammatory response during asthma. The activation of DP2 receptor pathway has a potential downstream effect on the pathophysiology of asthma. The binding of PGD2 with DP2 receptor can not only induce the activation of inflammatory pathway, but also amplify the effect of inflammatory pathway. In view of the fact that DP2 receptor is widely distributed in immune cells and structural cells involved in asthma, it is being explored as a new therapeutic target.

Alternative Names

PGD2 ELISA Kit

References

1. Athari SS. Targeting cell signaling in allergic asthma. Signal Transduct Target Ther. 2019 Oct 18;4:45.
2. Brightling CE, et al. The impact of the prostaglandin D2 receptor 2 and its downstream effects on the pathophysiology of asthma. Allergy. 2020 Apr;75(4):761-768.

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References

The prostaglandin D2 antagonist asapiprant ameliorates clinical severity in young hosts infected with invasive Streptococcus pneumoniae

Infect Immun

Authors: Bhalla M, Herring S, Lenhard A, Wheeler JR, Aswad F, Klumpp K, Rebo J, Wang Y, Wilhelmsen K, Fortney K, Bou Ghanem EN.

Abstract

Streptococcus pneumoniae (pneumococcus) remains a serious cause of pulmonary and systemic infections globally, and host-directed therapies are lacking. The aim of this study was to test the therapeutic efficacy of asapiprant, an inhibitor of prostaglandin D2 signaling, against pneumococcal infection. Treatment of young mice with asapiprant after pulmonary infection with invasive pneumococci significantly reduced systemic spread, disease severity, and host death. Protection was specific against bacterial dissemination from the lung to the blood but had no effect on pulmonary bacterial burden. Asapiprant-treated mice had enhanced antimicrobial activity in circulating neutrophils, elevated levels of reactive oxygen species (ROS) in lung macrophages/monocytes, and improved pulmonary barrier integrity indicated by significantly reduced diffusion of fluorescein isothiocyanate (FITC)-dextran from lungs into the circulation. These findings suggest that asapiprant protects the host against pneumococcal dissemination by enhancing the antimicrobial activity of immune cells and maintaining epithelial/endothelial barrier integrity in the lungs.

The Prostaglandin D2 Receptor CRTH2 Contributes to Airway Hyperresponsiveness during Airway Inflammation Induced by Sensitization without an Adjuvant in Mice

Int Arch Allergy Immunol

Authors: Hanzawa S, Sugiura M, Nakae S, Masuo M, Morita H, Matsumoto K, Takeda K, Okumura K, Nakamura M, Ohno T, Miyazaki Y.

Abstract

Introduction: Prostaglandin D2 (PGD2), which is produced mainly by Th2 cells and mast cells, promotes a type-2 immune response by activating Th2 cells, mast cells, eosinophils, and group 2 innate lymphoid cells (ILC2s) via its receptor, chemoattractant receptor-homologous molecules on Th2 cells (CRTH2). However, the role of CRTH2 in models of airway inflammation induced by sensitization without adjuvants, in which both IgE and mast cells may play major roles, remain unclear.

Methods: Wild-type (WT) and CRTH2-knockout (KO) mice were sensitized with ovalbumin (OVA) without an adjuvant and then challenged intranasally with OVA. Airway inflammation was assessed based on airway hyperresponsiveness (AHR), lung histology, number of leukocytes, and levels of type-2 cytokines in the bronchoalveolar lavage fluid (BALF).

Results: AHR was significantly reduced after OVA challenge in CRTH2 KO mice compared to WT mice. The number of eosinophils, levels of type-2 cytokines (IL-4, IL-5, and IL-13) in BALF, and IgE concentration in serum were decreased in CRTH2 KO mice compared to WT mice. However, lung histological changes were comparable between WT and CRTH2 KO mice.

Conclusion: CRTH2 is responsible for the development of asthma responses in a mouse model of airway inflammation that features prominent involvement of both IgE and mast cells.