Description
Introduction: Secondary Lymphoid-tissueChemokine (SLC) is a recently identified CC chemokine that is constitutively expressed in various lymphoid tissues and is a potent and specific chemoattractant for lymphocytes. The SLC gene and the gene encoding another lymphocyte-specific CC chemokine, EBI1-ligand chemokine (ELC), form a mini-cluster at human chromosome 9p13. Here, we show that SLC is a high affinity functional ligand for chemokine receptor 7 (CCR7) that is expressed on T and B lymphocytes and a known receptor for ELC. SLC induced a vigorous calcium mobilization in murine L1.2 cells stably expressing human CCR7. SLC tagged with the secreted form of alkaline phosphatase (SLC-SEAP) showed specific binding to CCR7 that was fully competed by SLC with an IC50 of 0.5 nm. SLC also induced a vigorous chemotactic response in CCR7-expressing L1.2 cells with a typical bell-shaped dose-response curve and a maximal migration at 10 nm. When assessed using CCR7-transfected L1.2 cells, SLC and ELC were essentially equivalent in terms of cross desensitization in calcium mobilization via CCR7, cross-competition in binding to CCR7, and induction of chemotaxis via CCR7. SLC and ELC were also shown to fully share receptors expressed on cultured normal T cells known to express CCR7. Notably, however, SLC was somehow less efficient in cross-desensitization against ELC in calcium mobilization and in cross-competition with ELC for binding when assessed using cultured normal T cells. Thus, SLC and ELC, even though sharing only 32% amino acid identity, constitute a genetically and functionally highly related subgroup of CC chemokines.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to SLC. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for SLC and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain SLC, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of SLC in the samples is then determined by comparing the O.D. of the samples to the standard curve