Human Progranulin ELISA Kit

Sample

Serum and plasma

Species Reactivity

Human

Detection Method

sELISA

Intended Use

The Human Progranulin ELISA Kit is intended to be used for quantitative measurement of human progranulin in human serum and plasma samples for research use.
Allow measurements in cell culture media too and in
specimens others than serum e.g. in Cerebrospinal fluid, Amnion fluid, Saliva, Urine, Breast milk.

Contents of Kit

1. Microtiter plate: ready for use, coated with human Progranulin antibody. Wells are separately breakable. (8x12) wells
2. Standards (A-E): lyophilized, contain recombinant Progranulin. Standard values are between 0.075 - 2.5 ng/ml (75, 250, 750, 1500 und 2500 pg/ml) Progranulin and have to be reconstituted with 1 ml (each) Dilution Buffer VP. Use 50 μl pro well in the assay.
3. Dilution Buffer VP: ready for use. Please use this for the reconstitution of Standards and Control Sera and for the dilution of Control Sera and Samples. Please shake before use! 1 x 50 mL
4. Control Sera KS1 and KS2: lyophilized, contain human Serum and should be reconstituted in each 250 μl Dilution Buffer VP. The Progranulin target values and the respective ranges are given on the certificate. The dilution should be according to the dilution of the respected samples. Use 50 μl per well in the assay. 1 x 250 μL
5. Antibody Conjugate (AK): ready for use, contains the biotinylated anti-Progranulin antibody. Use 50 μl for each well in the assay. 1 x 6 mL
6. Enzyme Conjugate (EK): contains HRP (Horseradish-Peroxidase)-labeled Streptavidin. Use 100 μl for each well in the assay. 1 x 12 mL
7. Washing Buffer (WP): 20-fold concentrated solution. Washing Buffer (WP) has to be diluted 1:20 with distilled or demineralised water before use (e.g. add the complete contents of the flask (50 ml) into a graduated flask and fill up with A. dest. to 1000 ml). Attention: After dilution the Washing Buffer is only 4 weeks stable, dilute only according to requirements. 1 x 50 mL
8. Substrate (S): ready for use, horseradish-peroxidase-(HRP) substrate. 1 x 12 mL
9. Stopping Solution (SL): ready for use, 0.2 M sulfuric acid. 1 x 12 mL
10. Sealing Tape: for covering the microtiter plate. 2

Storage

Store the kit at 2-8°C after receipt until its expiry date. The lyophilized reagents should be stored at –20 °C after reconstitution. Avoid repeated thawing and freezing.
The shelf life of the components after initial opening is warranted for 4 weeks, store the unused strips and microtiter wells airtight together with the desiccant at 2-8°C in the clip-lock bag, use in the frame provided. The reconstituted components standards A-E and Control Sera KS1 and KS2 must be stored at –20°C (max. 4 weeks). For further use, thaw quickly but gently (avoid temperature increase above room temperature and avoid excessive vortexing). The 1:20 diluted Washing Buffer WP is 4 weeks stable at 2-8°C.

Precision

The inter and intra assay coefficients of variability are below 8.0 and 4.4 %, respectively.
Inter-Assay-Variation (results of 14 independent determinations)

Intra-Assay-Variation

Sensitivity

The analytical sensitivity of the assay yields 0.018 ng/ml (pg/ml; as 2x SD of zero standard in 19-fold determination).

General Description

Progranulin is also known as Granulin Epithelin Precursor, Proepithelin or Acrogranin. It is a 68.5 kDa protein, consisting of 593 amino acids (inclusive Signalpeptid), which appears in vivo in strongly glycosylated form and therefore has a size of approximately 90 kDa.
Progranulin has seven conserved domains, which are separated by linker sequences. By means of proteolytic cleavage, catalyzed by serine proteases like e.g. elastase, 6-25 kDa large fragments result, that are called Granulines or Epithelines. Progranulin is expressed and secreted in particular in strongly proliferating tissues such as adenoid tissue, spleen, skin epithelium, gastrointestinal mucous membranes, haematopoietic cells and in tumor cells. Until now no specific receptors, which would obtain the effect of Progranulin or the Granulines are known.
Progranulin seems to be a factor, which affects the wound healing positively. In case of skin lesions the expression is increased in ceratinocytes, in macrophages and in neutrophile cells. Progranulin affects the wound healing indirectly by activation of macrophages and stimulation of angiogenesis in the damaged tissue. The physiological effects of Progranulin and Granulines are oppositional. Progranulin can restrain TNFα mediated pro-inflammatory processes. On the other hand the Granulines seem to stimulate the secretion of pro-inflammatory cytokines. The influence of Progranulin on inflammatory processes could be shown also in arteriosclerotic plaques. Here Progranulin is expressed by smooth muscle cells and affects the migration of monocytes and smooth muscle cells. In the central nervous system Progranulin is expressed in microglia and neurons (in neocortical and hippocampal pyramid cells as well as in purkinje cells in the cerebellum).
On mRNA level a clear increase of Progranulin expression could be shown during infections or injuries of the CNS, for example in mucopolysaccharidosis type I and IIIB, in viral inflammations of CNS, in amyotrophic lateral sclerosis and in Alzheimer's disease. Beyond that Progranulin seems to be of relevance in the development of sex specific differences during pre- and postnatal development and also for the neural plasticity in adults.
Progranulin and Frontotemporal Dementia (FTD)
5-10 % of all dementias are of the frontotemporal form. A mutation in the gene for Progranulin (PGRN) could be shown in 5-10 % of the patients suffering FTD. Nearly all pathological mutations lead to a premature transcription interruption and to rapid degradation of the mutated mRNA. This results in a PGRN haploinsufficiency with clearly decreased Progranulin concentrations in serum. Due to these results several studies were accomplished, in order to clarify the suitability of Progranulin as marker for the PRGN dependent frontotemporal dementia. The results of these studies show that Progranulin can detect already presymptomatically a FTD. Due to the missing standardisation and the use of different antibodies in the commercially available test systems cut off value must be evaluated for each assay separately.
Progranulin and Adiposity
Inflammatory processes are often increased in case of adiposity and type 2 diabetes, which is reflected by e.g. in the increase of the C-reactive Protein and pro-inflammatory cytokines e.g. IL-6. Youn et al. compared different groups of patients and have shown that the plasma concentration of Progranulin is significantly (1.4-fold) increased in type 2 diabetics compared to glucose-tolerant patients. The authors refer in particular to the positive correlation of the Progranulin concentration to the volume of the visceral adipose tissue. On the other hand no difference between slim and subcutaneous adipose patients has been detected in this study. For this reason the increase of the Progranulin concentration may reflect the body distribution of adipose tissue and thus represent a biomarker for visceral adipose tissue.
The Human Progranulin ELISA Kit is based on monoclonal antibodies, which detect with high specificity only Progranulin and not the single Granulines. Thus, a tool is available for the further investigation and validation of Progranulin as a biomarker for the visceral adipose tissue.

Standard Curve

The exemplary shown standard curve in Figure below cannot be used for calculation of your test results. You have to establish a standard curve for each test you conduct!