TLR4 ELISA Kit

Catalog No. ABIN6574197

Product Details TLR4 ELISA Kit

Target: TLR4 (Toll-Like Receptor 4 (TLR4))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.31 ng/mL - 20 ng/mL

Minimum Detection Limit: 0.31 ng/mL

Application: ELISA

Purpose: The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of TLR4 in human tissue homogenates, cell lysates, cell culture supernates.

We offer validation data (WB) for the kit components. So you can be sure to order a reliable ELISA kit product composed of high quality reagents.

Sample Type: Cell Culture Supernatant, Cell Lysate, Tissue Homogenate

Analytical Method: Quantitative

Specificity: This assay has high sensitivity and excellent specificity for detection of Toll Like Receptor 4 (TLR4)

Cross-Reactivity (Details): No significant cross-reactivity or interference between Toll Like Receptor 4 (TLR4) and analogues was observed.

Sensitivity: 0.118 ng/mL

Components:

• Pre-coated, ready to use 96-well strip plate, flat buttom
• Plate sealer for 96 wells
• Reference Standard
• Standard Diluent
• Detection Reagent A
• Detection Reagent B
• Assay Diluent A
• Assay Diluent B
• Reagent Diluent (if Detection Reagent is lyophilized)
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

Application Details

Comment:

Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.

Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.

Information on antibodies:
The provided antibodies and their host vary in different kits.

Sample Volume: 100 μL

Assay Time: 3 h

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and standards,
2. Add 100μL standard or sample to each well. Incubate 1 hours at 37 °C,
3. Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37 °C,
4. Aspirate and wash 3 times,
5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
6. Aspirate and wash 5 times,
7. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
8. Add 50μL Stop Solution. Read at 450nm immediately.

Reagent Preparation:

1. Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
2. Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 80 ng/mL. Firstly dilute the stock solution to 20 ng/mL and the diluted standard serves as the highest standard (20 ng/mL). Then prepare 7 tubes containing 0.5 mL Standard Diluent and use the diluted standard to produce a double dilution series. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL, and the last microcentrifuge tube with Standard Diluent is the blank as 0 ng/mL.
3. Detection Reagent A and Detection Reagent B - If lyophilized reconstitute the Detection Reagent A with 150μL of Reagent Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively.
4. Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:

1. Making serial dilution in the wells directly is not permitted.
2. Prepare standards within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for one pipetting.
4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
6. Contaminated water or container for reagent preparation will influence the detection result.

Sample Preparation:

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturationmay occur in these samples, leading to false results. Samples should therefore be stored for a short periodat 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thawcycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged toremove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determinecompatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibilityof causing a deviation due to the introduced chemical substance.The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in therange of the standard curve, the optimal sample dilution for the particular experiment has to be determined.Samples should then be diluted with PBS (pH =7.0-7.2).

Assay Precision: Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level of target were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level of target were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV < 10%
Inter-Assay: CV < 12%

Restrictions: For Research Use only

Handling

Precaution of Use: The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Storage: 4 °C/-20 °C

Storage Comment:

1. For unopened kit: All reagents should be stored according to the labels on the vials. The Standard, Detection Reagent A, Detection Reagent B, and 96-well Strip Plate should be stored at -20 °C upon receipt, while the other reagents should be stored at 4 °C.
2. For opened kits: the remaining reagents must be stored according to the above storage conditions. In addition, please return the unused wells to the foil pouch containing the desiccant and seal the foil pouch with the zipper.

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Expiry Date: 6 months

References for TLR4 ELISA Kit (ABIN6574197)

Hossain, Morandi, Tanasescu, Frakich, Caldano, Onion, Faraj, Erridge, Gran: "The Soluble Form of Toll-Like Receptor 2 Is Elevated in Serum of Multiple Sclerosis Patients: A Novel Potential Disease Biomarker." in: Frontiers in immunology, Vol. 9, pp. 457, (2019) (PubMed).

Ping, Xiaomu, Xufang, Liang: "Vinpocetine regulates levels of circulating TLRs in Parkinson's disease patients." in: Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology, Vol. 40, Issue 1, pp. 113-120, (2019) (PubMed).

Yan, Li, Zhu, Gao, Li, Zhang: "Increased TLR4 and TREM-1 expression on monocytes and neutrophils in preterm birth: further evidence of a proinflammatory state." in: The journal of maternal-fetal & neonatal medicine, pp. 1-9, (2018) (PubMed).

Li, Huang, Wang, Zhang: "Increased serum levels of high mobility group protein B1 and calprotectin in pre-eclampsia." in: International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics, Vol. 142, Issue 1, pp. 37-41, (2018) (PubMed).

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du Plessis, Korf, van Pelt, Windmolders, Vander Elst, Verrijken, Hubens, Van Gaal, Cassiman, Nevens, Francque, van der Merwe: "Pro-Inflammatory Cytokines but Not Endotoxin-Related Parameters Associate with Disease Severity in Patients with NAFLD." in: PLoS ONE, Vol. 11, Issue 12, pp. e0166048, (2017) (PubMed).

Liang, Aiqun, Jiwu, Ping: "TLR3 and TLR4 as potential clinical biomarkers for in-stent restenosis in drug-eluting stents patients." in: Immunologic research, Vol. 64, Issue 2, pp. 424-30, (2016) (PubMed).

Weijma, Vos, Ten Oever, Van Schilfgaarde, Dijksman, Van Der Ven, Van Den Berk, Brinkman, Frissen, Leyte, Schouten, Netea, Blok: "The Effect of Rosuvastatin on Markers of Immune Activation in Treatment-Naive Human Immunodeficiency Virus-Patients." in: Open forum infectious diseases, Vol. 3, Issue 1, pp. ofv201, (2016) (PubMed).

Sokół, Wąsik, Jankowski, Hołysz, Więckowska, Jagodziński: "Soluble Toll-Like Receptors 2 and 4 in Cerebrospinal Fluid of Patients with Acute Hydrocephalus following Aneurysmal Subarachnoid Haemorrhage." in: PLoS ONE, Vol. 11, Issue 5, pp. e0156171, (2016) (PubMed).

Mothapo, Ten Oever, Koopmans, Stelma, Burm, Bajramovic, Verbeek, Rikkert, Netea, Koopman, van der Ven: "Soluble TLR2 and 4 concentrations in cerebrospinal fluid in HIV/SIV-related neuropathological conditions." in: Journal of neurovirology, Vol. 23, Issue 2, pp. 250-259, (2016) (PubMed).

Ten Oever, Kox, van de Veerdonk, Mothapo, Slavcovici, Jansen, Tweehuysen, Giamarellos-Bourboulis, Schneeberger, Wever, Stoffels, Simon, van der Meer, Johnson, Kullberg, Pickkers, Pachot, Joosten et al.: "The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases. ..." in: BMC immunology, Vol. 15, pp. 55, (2015) (PubMed).

Pastorelli, Dozio, Pisani, Boscolo-Anzoletti, Vianello, Munizio, Spina, Tontini, Peyvandi, Corsi Romanelli, Vecchi: "Procoagulatory state in inflammatory bowel diseases is promoted by impaired intestinal barrier function." in: Gastroenterology research and practice, Vol. 2015, pp. 189341, (2015) (PubMed).

Gangarapu, Ince, Baysal, Kayar, Kılıç, Gök, Uysal, Şenturk: "Efficacy of rifaximin on circulating endotoxins and cytokines in patients with nonalcoholic fatty liver disease." in: European journal of gastroenterology & hepatology, Vol. 27, Issue 7, pp. 840-5, (2015) (PubMed).

Galliera, Drago, Vassena, Romanò, Gioia Marazzi, Salcito, Corsi Romanelli: "Toll-like receptor 2 in serum: a potential diagnostic marker of prosthetic joint infection?" in: Journal of clinical microbiology, Vol. 52, Issue 2, pp. 620-3, (2014) (PubMed).

Mastalerz-Migas, Pokorski, Kili?-Pstrusi?ska, Doskocz, Sapilak, Brydak: "Cytokines and toll-like receptors in the immune response to influenza vaccination." in: Advances in experimental medicine and biology, Vol. 836, pp. 35-40, (2014) (PubMed).

Yakut, Örmeci, Erdal, Keskin, Karayel, Tutkak, Soykan et al.: "The association between precancerous gastric lesions and serum pepsinogens, serum gastrin, vascular endothelial growth factor, serum interleukin-1 Beta, serum toll-like receptor-4 levels and ..." in: Clinics and research in hepatology and gastroenterology, Vol. 37, Issue 3, pp. 302-11, (2013) (PubMed).

Kacerovsky, Andrys, Hornychova, Pliskova, Lancz, Musilova, Drahosova, Bolehovska, Tambor, Jacobsson: "Amniotic fluid soluble Toll-like receptor 4 in pregnancies complicated by preterm prelabor rupture of the membranes." in: The journal of maternal-fetal & neonatal medicine, Vol. 25, Issue 7, pp. 1148-55, (2012) (PubMed).

Yang, Xie, Deng, Qin: "Expression of soluble Toll-like receptors in pleural effusions." in: Chinese medical journal, Vol. 123, Issue 16, pp. 2225-30, (2010) (PubMed).

Target Details for TLR4

Target: TLR4 (Toll-Like Receptor 4 (TLR4))

Alternative Name: Toll Like Receptor 4 (TLR4) (TLR4 Products)

Synonyms: ARMD10 ELISA Kit, CD284 ELISA Kit, TLR-4 ELISA Kit, TOLL ELISA Kit, Lps ELISA Kit, Ly87 ELISA Kit, Ran/M1 ELISA Kit, Rasl2-8 ELISA Kit, toll like receptor 4 ELISA Kit, toll-like receptor 4 ELISA Kit, TLR4 ELISA Kit, Tlr4 ELISA Kit

UniProt: O00206

Pathways: TLR Signaling, Activation of Innate immune Response, Cellular Response to Molecule of Bacterial Origin, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Toll-Like Receptors Cascades, Inflammasome, S100 Proteins