Sample
Serum, Plasma, WBH and Mucus
Intended Use
The Multi Species Vitellogenin ELISA Kit is a sensitive enzyme linked immunosorbent assay for the quantitative determination of vitellogenin (VTG) in fish serum, plasma whole body homogenate (WBH) and mucus
Contents of Kit
1. 96-well plate coated with mVTG Antibody 1 plate, 12 break apart strips of 8 wells (12 x 8 in total), in a frame.
Ready to use.
2. Standard Stock lyophilized 2 x. 105 ng
3. Control C1 lyophilized 2 x. Concentration see Certificate of Analysis.
4. Control C2 lyophilized 2 x. Concentration see Certificate of Analysis.
5. Wash Buffer 50x. 1 x 30 ml. Dilute 1:50 with deionized water.
6. Dilution Buffer 1 x 55 ml. Ready to use.
7. Matrix Solution 1 x 7 ml. Ready to use.
8. Biotinylated Antibody (Biotin-AB) 1 x 12 ml. Ready to use.
9. Streptavidin Peroxidase Conjugate (SA- HRP Conj.) 1 x 12 ml. Ready to use.
10. TMB Substrate 1 x 12 ml. Ready to use.
11. Stop Solution – 1 M HCI 1 x 12 ml. 1 M hydrochloric acid, ready to use.
12. Kit instruction 1 x.
Storage
The kit has to be stored at 2-8 °C until expiry date. Do not freeze. Store unused reagents at 2-8 °C.
Detection Range
2.6 - 210 ng/mL
Sensitivity
LLOQ < 2.6 ng/ml
LLD 0.7 ng/ml
General Description
In oviparous animals, vitellogenin (VTG) is an estrogen induced yolk precursor protein mainly synthesized in the liver to be deposited in the maturing oocytes, where it is split in the yolk proteins lipovitellin 1, lipovitellin 2 and phosvitin. These yolk proteins serve as nourishment storage for the developing embryos. Nonphysiological induction of vitellogenin in males or in juvenile fish is thought to indicate an estrogen mediated endocrine disruption. Therefore VTG determination is one of the core endpoints in screening and testing for endocrine disrupting chemicals standardized in the OECD Guidelines for the testing of chemicals for estrogenic activity.
Normally vitellogenin is measured in blood samples or whole body homogenate (WBH) - both sample types require invasive and destructive treatment of the fish. Blood is difficult to collect, in particular where very small fish are concerned, or in approaches where the animals must survive sampling. This is particularly important in field monitoring in order to avoid impact on the population under investigation.
Recently, several cell types have been shown to produce VTG after estrogen stimulation, including those of the epidermal mucosa. Further studies showed that both VTG and estrogen receptor genes are expressed in epidermal cells. Immunoaffinity and mass fingerprint analysis showed induction of identical VTG peptides in liver and epidermis. VTG contents in the serum demonstrated a similar dose-response pattern in the epidermis and the blood. Even though the VTG concentration in the skin mucus is an order of magnitude lower than in blood serum or in body homogenates (containing liver tissue), the skin mucosa is very well suited as a matrix to determine exogenous VTG induction caused by environmental chemicals with affinity to estrogen receptors. By using a highly sensitive ELISA in combination with an unique sampling and extraction system the determination of mucosa born VTG determination has the following advantages:
• Simple and highly standardized sampling technique and sample preparation.
• Strictly defined matrix without protease contamination caused by non-target tissues or lymphatic fluid.
• Non-destructive and thereby allowing several subsequent samplings in order to record a kinetic of VTG induction with a maximum known to appear within 1-2 weeks after exposure. Therefore mucosa tests are compatible with acute as well as chronical OECD test methods.
• Epithelial organized epidermis is directly exposed to exogenous estrogens and thereby allowing a direct comparison with in vitro test using estrogen sensitive vitellogenin producing fish cell lines.
• Lower degree of interference with endogenous VTG production (in females) and bio concentration or enterohepatic circulation of the effective estrogen (xenoestrogen) and thereby showing a clear dose response relationship.
• Stability of standards and samples if prescribed storage conditions are observed.
Citations
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