PSGL-1 ELISA Kit

Sample

cell culture supernatant, serum, plasma, other body fluids

Species Reactivity

Human

Intended Use

The human PSGL-1 ELISA is an enzyme-linked immunosorbent assay for the quantitative detection of human PSGL-1. The human PSGL-1 ELISA is for research use only. Not for diagnostic or therapeutic procedures.

Contents of Kit

1. 1 aluminium pouch with a Microwell Plate coated with monoclonal antibody to human PSGL-1
2. 1 vial (70 μl) Biotin-Conjugate anti-human PSGL-1 monoclonal antibody
3. 1 vial (150 μl) Streptavidin-HRP
4. 2 vials human PSGL-1 Standard lyophilized, 100 U/ml upon reconstitution
5. 1 vial (50 ml) Sample Diluent
6. 1 vial (5 ml) Assay Buffer Concentrate 20x (PBS with 1% Tween 20, 10% BSA)
7. 1 bottle (50 ml) Wash Buffer Concentrate 20x (PBS with 1% Tween 20)
8. 1 vial (15 ml) Substrate Solution (tetramethyl-benzidine)
9. 1 vial (15 ml) Stop Solution (1M Phosphoric acid)
10. 4 Adhesive Films

Storage

Store kit reagents between 2° and 8°C. Immediately after use remaining reagents should be returned to cold storage (2 - 8°C).
Expiry of the kit and reagents is stated on labels.

The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling.

Precision

Intra-assay
Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of human PSGL-1. 2 standard curves were run on each plate. Data below show the mean human PSGL-1 concentration and the coefficient of variation for each sample (see Table 3). The calculated overall intra-assay coefficient of variation was 3.2%.
The mean human PSGL-1 concentration and the coefficient of variation for each sample.
Table 3

Inter-assay
Assay to assay reproducibility within one laboratory was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of human PSGL-1. 2 standard curves were run on each plate. Data below show the mean human PSGL-1 concentration and the coefficient of variation calculated on 18 determinations of each sample (see Table 4). The calculated overall inter-assay coefficient of variation was 6.6%.
The mean human PSGL-1 concentration and the coefficient of variation of each sample.
Table 4

Sensitivity

The limit of detection of human PSGL-1 defined as the analyte concentration resulting in an absorbance significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 1.0 U/ml (mean of 6 independent assays).

General Description

The family of selectins consists of three structurally and functionally related molecules. L-selectin is constitutively expressed on neutrophils, P-selectin is found on platelets and is stored in Weibel-Palade bodies from where it is transported to the cell surface upon endothelial activation. E-selectin is expressed on endothelial cells. Due to a common structural element, the aminoterminal lectin-like domain, the selectins are able to bind to carbohydrate ligands. Different putative ligand structures have been identified for which the selectins show high affinity. These structures include oligosaccharides, phophorylated saccharides, sulfopolysaccharides and lipids. It was shown that glycoproteins represent the biological relevant ligands for selectins. While the ligands for E- and L-selectins with primary binding activity have not been identified so far, the functionally most important ligand for P-selectin has been identified. The mucin-like glycoprotein PSGL-1 (P-selectin Glycoprotein Ligand-1) has been cloned and sequenced.
PSGL-1 has been shown to be a transmembrane protein which forms homodimers via disulfide bridges of two 120 kDa chains.
PSGL-1 is expressed on cells of myeloid, lymphoid and dendritic lineage. The binding of P-selectin is regulated by different degrees and forms of glycosylation. An interaction of L-selectin with PSGL-1 in the process of neutrophil aggregation has been shown. However, PSGL-1 does not seem to be the primary ligand for L-selectin.
Presently it is not known which cells, apart from leukocytes, express PSGL-1 and what role PSGL-1 plays on these cells. The metastic potential of the majority of cells which bind to P-selectin is however in close correlation with the functional expression of PSGL-1 on these cells.
The regulation of PSGL-1 is not yet well described. Glycosyltransferases sure play an important role in activation. The deactivation of PSGL-1 is so far unclear. The cleavage of the protein from the cell surface is one mechanism involved in the deactivation process.
Following this shedding, a soluble form of PSGL-1 is detectable in the circulation. This soluble isoform of PSGL-1 is still capable of binding to P-selectin, thus representing a competitor for cellular PSGL-1 through which regulation in many physiological and pathological processes can take place.