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Human CRTAM DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
4 hours 40 minutes (after plate preparation)
Sample Volume Required
100 µL
Assay Range
156.0 - 10,000 pg/mL
Sufficient Materials
For fifteen 96-well plates*
Specificity
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human CRTAM. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits

Kit Content

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008B) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4

Block Buffer: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered

Reagent Diluent: 0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline (20 mM Trizma base, 150 mM NaCI) pH 7.2-7.4, 0.2 μm filtered

Substrate Solution: TMB ELISA Substrate (Catalog # DY999B)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990)

Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

 

*For the Reagent Diluent and Blocking Buffer recommended for a specific DuoSet ELISA Development Kit, please see the product.

Data Example

Human CRTAM ELISA Standard Curve

Product Datasheets

Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: CRTAM

CRTAM (Class I-restricted T cell-associated molecule) is a nectin family member of the immunoglobulin superfamily that is expressed by activated CD8+ and NK T cells. NK activation receptor engagement, but not cytokines, also induce NK CRTAM expression. CRTAM is found in spleen, thymus, small intestine, peripheral blood, and in brain where it is highly expressed by Purkinje cells of the cerebellum.

Human CRTAM is a 393 amino acid (aa), 80 kDa type I transmembrane glycoprotein. CRTAM is a homodimer on the cell surface but does not show homotypic binding in trans. The high affinity of CRTAM/IGSF4 adhesion allows CRTAM to disrupt IGSF4 homotypic interactions. IGSF4 and T cell receptor co-engagement of CRTAM-expressing CD8+ cells induces increased IFN-gamma or IL-22 production. A role in cancer surveillance through NK cell-mediated rejection of IGSF4-expressing tumors has been proposed. IGSF4 is expressed broadly, including on epithelia, neurons, a subset of tonsillar B cells and a rare splenic T zone-restricted BCDA3+ dendritic cell population which interacts with CRTAM.

Long Name:
Class I-restricted T-cell-associated Molecule, Nectin
Entrez Gene IDs:
56253 (Human); 54698 (Mouse); 102125896 (Cynomolgus Monkey)
Alternate Names:
CD355 antigen; CD355; class I MHC restricted T cell associated molecule; class-I MHC-restricted T cell associated molecule; Class-I MHC-restricted T-cell-associated molecule; CRTAM; cytotoxic and regulatory T cell molecule; cytotoxic and regulatory T-cell molecule

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

FAQs

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