Human Vaspin ELISA Kit

Sample

Serum

Species Reactivity

Human

Detection Method

sELISA

Intended Use

The Human Vaspin ELISA Kit is intended to be used for quantitative measurement of human Vaspin in human serum for research purposes.

Contents of Kit

1. Microtiter plate: ready for use, coated with rabbit-anti-hVaspin-antibody. Wells are separately breakable. (8x12) wells
2. Standards (A-E): lyophilized, (human Vaspin), concentrations are given on the QC-certificate. 5 x 750 μL
3. Control Serum 1 (KS1): lyophilized, (human serum), concentration is given on the QC-certificate. 1 x 250 μL
4. Control Serum 2 (KS2): lyophilized, (human serum), concentration is given on the QC-certificate. 1 x 250 μL
5. Antibody Conjugate (AK): ready for use, contains rabbit anti-hVaspin antibody, biotinylated. 1 x 12 mL
6. Enzyme Conjugate (EK): ready for use, contains horseradish-peroxidase conjugated to streptavidin. 1 x 12 mL
7. Dilution Buffer (VP): ready for use. Please shake before use! 1 x 120 mL
8. Washing Buffer (WB): 20-fold concentrated solution. 1 x 50 mL
9. Substrate (S): ready for use, horseradish-peroxidase-(HRP) substrate, stabilised H₂O₂ Tetramethylbencidine. 1 x 12 mL
10. Stopping Solution (SL): ready for use, 0.2 M sulfuric acid. 1 x 12 mL
11. Sealing Tape: for covering the microtiter plate. 3

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 WB is 4 weeks stable at 2-8°C

Precision

Intra-Assay Variance
Three exemplary serum samples were diluted and measured 20 times in one assay.

Inter-Assay Variance
Serum samples were diluted as recommended and Vaspin concentration was measured in various independent tests.

Sensitivity

We measured the blank (dilution buffer only) 16 times in one assay. The resulting standard deviation was used for calculating the concentration which can be differentiated from the blank. Thus, lowest amount of Vaspin detectable is 4 pg/mL.

General Description

The Human Vaspin ELISA Kit is based on polyclonal rabbit antisera raised by genetic immunization of the rabbits. Vaspin also known as SerpinA12 is a serine protease inhibitor and consists of 395 amino acids forming 3 β-sheets and 9 α-helices. Molecular weight of Vaspin is about 45.2 kDa. It does not form multimeric aggregates or intra-molecular disulfide bridges and no binding proteins in human serum are known. The Vaspin gene is not only expressed by subcutaneous and visceral adipose but also by liver tissue, in the pancreas and in the human epidermis (granular keratinocytes / GK cells).
"Serum Vaspin levels were highest in the early morning before breakfast and fell to trough levels within 2 h after breakfast. Serum Vaspin levels also showed a preprandial rise and postprandial fall at lunch and dinner, although at lesser degrees than at breakfast. Intermeal Vaspin concentrations reached a nadir in the mid-afternoon and showed a nocturnal rise, with peak nighttime Vaspin levels being approximately 250 % of nadir levels. Unscheduled food ingestion after a prolonged fast significantly reduced serum Vaspin levels, suggesting that energy intake itself has a suppressive effect on serum Vaspin levels. The diurnal patterns of serum Vaspin concentrations were exactly reciprocal to that of insulin and of glucose".
A sexual dimorphism has been detected with higher Vaspin levels in girls increasing with age and pubertal stage. A preliminary investigation (n = 81) of Vaspin levels in healthy adult blood donors revealed higher Vaspin levels in women decreasing with increasing age. In this context, it is important to reflect, that oral contraceptives significantly increase serum Vaspin concentration.
Serum Vaspin concentration is independent of BMI but negatively associated with insulin sensitivity and obesity, thus "Vaspin was increased with worsening insulin resistance". If glucose metabolism / insulin sensitivity is improved by therapeutic intervention e.g. rosiglitazone, plasma Vaspin concentration decreases significantly. Interestingly lifestyle intervention results in increasing adiponectin concentrations as well as in improved insulin sensitivity but after a 10 month intervention Vaspin concentration remain unchanged. Thus, the mechanism regulating the Vaspin concentration in circulation is still unclear. Vaspin concentration might be even more influenced by glucose uptake than by body fat at least in pre-pubertal children.
In insulin resistance, diabetes as well as atherosclerosis inflammatory processes are involved and Vaspin might be a link between the endocrine and the immune system. To elucidate the role of Vaspin in inflammation its influence on TNF-α-stimulated production of reactive oxygen species was investigated in smooth muscle cells. Vaspin significantly decreased the TNF-α induced monocyte adhesion to SMCs as well as TNF-α induced intracellular signal cascade.
The diagnostic value of Vaspin remains unclear, conflicting results question its value as biomarker for visceral or total adipose tissue. As well as regarding insulin resistance while in children Vaspin might correlate with insulin sensitivity but in adults no correlation was found.
The Human Vaspin ELISA Kit is a tool for the further investigation and validation of Vaspin as a biomarker for the visceral adipose tissue, insulin sensitivity and glucose tolerance.

Standard Curve

The following data is for demonstration only and cannot be used in place of data generation at the time of assay.

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!