The TECO® REACH Medaka Vitellogenin ELISA kit is a sensitive enzyme linked immunosorbent assay for the quantitative determination of vitellogenin in serum, whole body homgenate (WBH) and mucus of cyprinids according to EC regulation Nr. 440/2008 (REACH) from July 10th 2015/ Document D039048/03.
Vitellogenin determination is one of the core endpoints in screening and testing for endocrine disrupting chemicals standardized in the OECD Guidlines for the testing of chemicals for estrogenic activity:
- OECD (2009), Test No.229
- OECD (2009), Test No.230
- OECD (2011), Test No.234
REACH kits meet strict quality assurance regulations by including, in addition to the standard ELISA materials, the following components in the kit:
- 1 break-apart strip of 8 wells for Non-Specific Binding (NSB) assessment.
- An extra inter-assay reference Standard Stock from a different vitellogenin ELISA production lot.
- A package insert that includes the specific requirements for vitellogenin testing within REACH regulations.
Vitellogenin determination is useful in ecotoxicological studies to determine the presence or effect of estrogenic compounds in the water.
Assay Measuring Range: 2.6-210 ng/ml
Assay Sensitivity: < 0.1 ng/ml
Incubation time: 4.0 hours
Sample Volume: 50 µl
- Serum: Store fresh serum samples immediately after collection at -20°C or lower until assayed.
- WBH: Store fresh WBH samples immediately after collection at -20°C or lower until assayed.
- Mucus: Collect as described in the TECO® Mucus Collection Set (TE1034). Mucus containing swabs can be stored several months at <-20°C.
- Serum levels are in the range of µg/ml up to mg/ml
- WBH levels are in the range of mg/ml
- Mucus level are in the range of ng/ml
- Antibody Coated Microassay Plate: 96-well plate (12×8 break-apart well strips coated with IgG directed against Medaka VTG)
- Non-Specific Binding (NSB) Wells: 1 break-apart strip of 8 wells coated without specific binding antibodies
- Standard Stock Solution: 105 ng, 2 vials
- Control C1: low control, 2 vials
- Control C2: high control, 2 vials
- Inter-Assay Reference Standard: 15.0 ng, 1 vial
- Wash Buffer: 1 x 30 ml, 50X (Dilute 1:50 with deionized water.)
- Dilution Buffer: 1 x 55 ml. Ready to use.
- Matrix Solution: 1 x 7 ml. Ready to use.
- Biotinylated Antibody (Biotin-AB): 1 x 12 ml. Ready to use.
- Streptavidin Peroxidase Conjugate (SA- HRP Conj.): 1 x 12 ml. Ready to use.
- TMB Substrate: 1 x 12 ml. Ready to use.
- Stop Solution: 1 x 12 ml 1M HCl. Ready to use.
Materials required but not supplied:
• Pipettes, 10 μl – 1000 μl
• Multichannel pipettes , 50 μl – 100 μl
• Graduated cylinders for reconstituting and diluting reagents
• Manual Aspiration System or automatic washer for ELISA plates
• Distilled water
• Vortex mixer
• ELISA plate reader suitable for 96 well formats and capable of measuring at 450 nm (Reference: 590-650 nm).
• ELISA plate shaker (500 rpm)
For mucus samples: The Mucus Collection Set (TE1034), which includes Extraction Buffer and validated Sampling Swabs is also required.
The TECO® REACH Medaka Vitellogenin ELISA kit is a 96-well immuno-capture ELISA product. Serum, WBH and mucus samples are incubated with the vitellogenin specific antibody-coated microtiter plate. After unbound material is washed out, a polyclonal biotinylated antibody binds to the vitellogenin. In the following incubation step, a streptavidin- peroxidase conjugate binds to the biotinylated antibody. In the final substrate reaction, the color development
is directly proportional to the amount of vitellogenin in the sample.
The standard range of the assay is between 0 and 210 ng/ml. A calibration curve can be established by plotting standard concentration on the x-axis (linear scale) against the absorbance of the standards on the y-axis (linear scale). The vitellogenin concentrations in samples can then be read off the calibration curve. A 4-parameter curve fit should be used for automatic data reduction. If samples were pre-diluted, the concentration can be obtained by multiplying the value read off the calibration curve by the dilution factor. A dilution correction for mucus is not necessary if only the 0.5 ml Extraction Buffer was added to the swab. Samples with higher absorbance values than Standard A should be tested again, pre-diluted with Dilution Buffer, and then this additional dilution should be taken in account for the concentration calculation.
TECO® REACH Medaka VTG ELISA Standard Curve Example
- Medaka, Japanese rice fish (Oryzias latipes)
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. Non-physiological 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 can be difficult to collect, in particular where very small fish are concerned and where the animals must survive sampling.
Recently, several cell types have been shown to produce VTG after estrogen stimulation, including those of the epidermal mucosa. Even though the VTG concentration in the skin mucus is an order of magnitude lower than in blood, serum or body homogenates (containing liver tissue), the skin mucosa is very well suited as a matrix for determining exogenous VTG induction caused by environmental chemicals with affinity to estrogen receptors. By using a sensitive ELISA in combination with a 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, thereby allowing several subsequent samplings in order to record a kinetic of VTG induction. A maximum is known to appear after 7 days of exposure. Therefore, Mucosa tests are fully 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.
The TECO® REACH Medaka Vitellogenin ELISA kit allows for testing samples of blood, homogenate and/or mucus. These sample types provide researchers with good sampling options especially when conducting time-course studies or field research where it is critical to preserve the life of the fish.