Human TSLP Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human TSLP immunoassay is a 4.5 hour sandwich-type solid phase ELISA designed to measure human TSLP in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human TSLP and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human TSLP showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human TSLP.

Preparation and Storage

Background: TSLP

Thymic Stromal Lymphopoietin (TSLP) was originally identified as an activity from the conditioned medium of a mouse thymic stromal cell line that promoted the development of B cells. The activities of mouse TSLP overlap with, but are distinct from, those of mouse IL-7. Both mouse TSLP and IL-7 can co-stimulate growth of thymocytes and mature T cells, and support B lymphopoiesis in long-term cultures of fetal liver cells and bone-marrow cells. Whereas mouse IL-7 facilitates the development of B220⁺/IgM^- pre-B cells, mouse TSLP promotes the development B220⁺/IgM⁺ B cells. Human TSLP was reported to preferentially stimulate myeloid cells, inducing the release of T cell-attracting chemokines from monocytes and enhancing the maturation of CD11c⁺ dendritic cells. Human TSLP cDNA encodes a 159 amino acid (aa) residue precursor protein with a 28 aa signal sequence. Within the mature region, six of the seven cysteine residues present in the mouse TSLP involved in intramolecular disulfide bond formation are conserved in the human TSLP. Human TSLP shares approximately 43% aa sequence identity with mouse TSLP. By Northern blot analysis, human TSLP expression has been detected in many tissues with the highest expressions in heart, liver, testis and prostate. TSLP signals through a heterodimeric receptor complex that consists of IL-7 R alpha and the TSLP R, a member of the hemopoietin receptor family most closely related to Rgamma_c.

TSLP R, also named Delta and CRLM-2 (Cytokine Receptor-like Module-2), was originally cloned as a novel type 1 cytokine receptor with similarity to the common gamma chain (gamma c). It was subsequently identified as a subunit of the cellular receptor for the IL-7-like cytokine TSLP and termed TSLP R. TSLP R shows 24% sequence identity with the mouse gc receptor. TSLP R expression is constitutive in immune and hematopoietic cells, but is up-regulated in Th2 cells.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.