CD40 Ligand ELISA Kit

Produktnummer ABIN1029800

Dieses Colorimetric ELISA kit dient der quantitativen Messung von Human CD40 Ligand.

Kurzübersicht für CD40 Ligand ELISA Kit (ABIN1029800)

Target: CD40 Ligand (CD40LG)

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 0.156 ng/mL - 10 ng/mL

Applikation: ELISA

Proben: Cell Lysate, Plasma, Serum, Tissue Homogenate

Produktdetails CD40 Ligand ELISA Kit

Untere Nachweisgrenze: 0.156 ng/mL

Verwendungszweck: The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of CD40L in human serum, plasma, tissue homogenates, cell lysates and other biological fluids.

Analytische Methode: Quantitative

Spezifität: This assay has high sensitivity and excellent specificity for detection of this index.

Kreuzreaktivität (Details): No significant cross-reactivity or interference between this index and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between this index and all the analogues, therefore, cross reaction may still exist.

Sensitivität: 0.051 ng/mL

Bestandteile:

• Pre-coated, ready to use 96-well strip plate
• Plate sealer for 96 wells
• Standard
• Diluents buffer
• Detection Reagent A
• Detection Reagent B
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

Benötigtes Material:

• Microplate reader with 450 ± 10nm filter.
• Precision single or multi-channel pipettes and disposable tips.
• Eppendorf Tubes for diluting samples.
• Deionized or distilled water.
• Absorbent paper for blotting the microtiter plate.
• Container for Wash Solution.

Anwendungsinformationen

Probenmenge: 100 μL

Testdauer: 1 - 4.5 h

Plattentyp: Pre-coated

Protokoll: 1. Prepare all reagents, samples and standards
2. Add 100µL standard or sample to each well. Incubate 2 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 1 hour at 37°C
6. Aspirate and wash 5 times
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37°C
8. Add 50µL Stop Solution. Read at 450nm immediately.

Probennahme: Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4 °C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2 - 8 °C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.02Mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000xg. Remove the supernate and assay immediately or aliquot and store at ≤-20 °C.
Cell Lysates - Cells must be lysed before assaying according to the following directions:""

• Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
• Wash cells three times in cold PBS.
• Resuspend cells in PBS (1x) and the cells was subject to ultrasonication for 4 times (or Freeze cells at≤-20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)
• Centrifuge at 1500xg for 10 minutes at 2 - 8 °C to remove cellular debris. Cell culture supernates and Other biological fluids - Centrifuge samples for 20 minutes at 1000xg. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C. Avoid repeated freeze/thaw cycles.

Aufbereitung der Proben:

1. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
5. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.

Testdurchführung:

1. Prepare all reagents, samples and standards.
2. Add 50μL standard or sample to each well. And then add 50μL prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37 °C.
3. Aspirate and wash 3 times#Add 100μL prepared Detection Reagent B. Incubate 1 hour at 37 °C.
4. Aspirate and wash 5 times.
5. Add 90μL Substrate Solution. Incubate 15-25 minutes at 37 °C.
6. Add 50μL Stop Solution. Read at 450 nm immediately.

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

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

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

Handhabung: Note: To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end..

Lagerung: 4 °C,-20 °C

Informationen zur Lagerung: The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C.Other reagents are kept according to the labels on vials. But for long term storage, please keep the whole kit at -20°C. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.

Haltbarkeit: 12 months

Details zu CD40 Ligand

Target: CD40 Ligand (CD40LG)

Andere Bezeichnung: CD40L

Hintergrund: Alternative Names: CD154, TRAP, HIGM1, IGM, IMD3, TBAM, T-BAM, TNFSF5, Gp39, TNF Superfamily Member 5, Hyper-IgM Syndrome, TNF-Related Activation Protein, T-Cell B-Cell Activating Molecule

Gen-ID: 959

UniProt: P29965

Pathways: NF-kappaB Signalweg, Production of Molecular Mediator of Immune Response, Cancer Immune Checkpoints