TECO® Perch (Perciformes) Vitellogenin ELISA

The Perch (Perciformes) Vitellogenin ELISA Kit is a sensitive sandwich enzyme linked immunosorbent assay for the quantitative determination of vitellogenin in serum and mucus of perciformes.

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

Vitellogenin determination is used in ecotoxicological studies to determine estrogenic compounds in the water.

Vitellogenin levels can be used to determine the sex and the status of sexual maturation of fish.

Studies

Stimulation Studies using estradiol (E2), Ethinyl-Estradiol (EE2,) Bisphenol A show clear increases in Vitellogenin levels in serum and mucus.

Range: 0-80 ng/ml

Sensitivity: < 0.5ng/ml

Incubation time: 4.0 hours

Sample volume: 50 µl

Sample preparation:

• Serum: Store fresh serum samples immediately after collection at
  -20°C or lower until assayed.
• Mucus: Collect mucus as described in the TECO® Mucus Collection Set (TE1034). Mucus-containing swabs can be stored several months at <-20°C

Reference values:

• Serum levels are in the range of µg/ml up to mg/ml
• Mucus level are in the range of ng/ml

Reagents

Materials required and not supplied

• Pipettes 10 μl – 1000 μl
• Multichannel pipettes for 50 μl – 100 μl
• Graduated cylinders for reconstituting or diluting reagents
• Manual Aspiration System or automatic washer for ELISA plates
• Aqua dest
• 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) (orbital shaker)
• Software package for data generation and analysis

Species

• Fish Perciformes
• Bluegill (Lepomis macrochirus)
• European Perch (Perca fluviatilis)

Assay Principle

The TECO® Perch Vitellogenin EIA Kit is a 96 well immuno-capture ELISA product. Mucus or serum 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.

Assay Procedure

All determinations (standards, controls and samples) should be assayed in duplicate. When performing the assay, the standards, controls and samples should be pipetted as fast as possible (<15 minutes).

To avoid distortions due to differences in incubation times, HRP conjugate, substrate solution and stop solution should be added to the plate in the same order and with the same time interval as the samples. A multichannel pipette is essential.

Allow all reagents to stand at room temperature (20–25 °C) for at least 30 minutes. During all incubation steps, plates should be sealed with the adhesive foil or a plastic cover. For light protection, incubate in a dark chamber or cover plate with aluminum foil.

1. Allocate the wells of the Microtiterplate  1  for standards, controls and samples.
2. Pipette 50 μl matrix solution  4  (multichannel pipette) into all wells.
3. Add 50 μl of each prepared standard ( A  –  F ), pre-diluted controls ( C1   and  C2 ) and samples into the corresponding wells.
4. Cover the wells and incubate the plate for 120 ± 5 min at room temperature (18–30 °C) on a shaker (500 rpm).
5. After incubation, aspirate the contents of the wells and wash 3 times with 350 μl diluted wash buffer  2 . The use of an automatic plate washer is recommended.
6. Following the last washing step, pipette 100 μl of the biotinylated AB  5  in each well (multichannel pipette).
7. Cover the wells and incubate the plate for 60 ± 5 min at room temperature (18–30 °C) on a shaker (500 rpm).
8. After incubation, wash the wells 3 times with wash buffer as described in step 5.
9. Following the last washing step, pipette 100 μl of the SA-HRP conjugate  6  in each well (multichannel pipette).
10. Cover the wells and incubate the plate for 30 ± 5 min at room temperature (18–30 °C) on a shaker (500 rpm).
11. After incubation, wash the wells 5 times with wash buffer as described in step 5.
12. Pipette 100 μl of the TMB substrate solution  7  in each well (multichannel pipette).
13. Incubate the plate for 15-30 min, in the dark, at room temperature (18–30 °C) on a shaker (500 rpm).
14. Stop the reaction by adding 100 μl of stop solution  8  (multichannel pipette).
15. Measure the color reaction within 10 minutes at 450 nm (reference filter between 590–650 nm). If the extinction of the Standard A (80 ng/ml) exceeds 3.0, the measurement may be repeated at 405 nm.

Background

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 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. 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
• Nondestructive and thereby allowing several subsequent samplings in order to record a kinetic of VTG induction with a maximum known to appear after 7 days of exposure. Therefor Mucosa test 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.