Introduction

Detection of the egg yolk precursor vitellogenin (Vtg) in blood and tissue samples of juvenile and male fish is a simple and sensitive biomarker for endocrine disrupting chemicals (EDCs) with oestrogenic effects (Arukwe & Goksøyr, 2003; Sumpter & Jobling, 1995). Measurement of Vtg has become an accepted routine screening test for oestrogenic and anti-androgenic effects of EDCs in fish. This Enzyme Linked Immunosorbent Assay (ELISA) can readily be combined with standard fish toxicology tests developed under the framework of governmental organizations, e.g. the OECD and the US EPA. This ELISA is developed for a standard test species used in many ecotoxicology laboratories throughout the world, the zebrafish (Danio rerio).

Safety Instruductions

For research use only. Not for human use or drug use. Not for clinical diagnostic use. These reagents contain sodium azide as preservative. Do not use internally or externally in humans or animals. The kit contains OPD (o-phenylenediamine) tablets. Since OPD is toxic and may be carcinogenic, contact should be avoided and gloves and suitable protective clothing should be used when handling tablets or solutions made from the tablets (see Material Safety Data Sheet [MSDS] for the OPD tablets for more details about proper handling and waste disposal). As all chemicals should be considered potentially hazardous, it is advisable to wear suitable protective clothing during handling of this kit. Avoid contact with skin and eyes

Storage and Stability

Store the kit at 2-8°C upon arrival. Do not freeze. See expiry date on the kit box for stability of the kit. Unused pre-coated microplates should be stored airtight with enclosed desiccant at 2-8°C.

Warranty and limitation of Remedy

Biosense Laboratories AS (hereafter: Biosense) makes no warranty of any kind, expressed or implied, including, but not limited to, the warranties of fitness for a particular purpose and merchantability, which extends beyond the description of the chemicals on the face hereof, except that the material will meet our specifications at the time of delivery. Buyer’s exclusive remedy and Biosense´s sole liability hereunder shall be limited to refund of the purchase price of, or at Biosense´s option, the replacement of, all material that does not meet our specifications. Biosense shall not be liable otherwise or for incidental or consequential damages, including, but not limited to, the costs of handling. Said refund or replacement is conditioned on Buyer giving written notice to Biosense within thirty (30) days after arrival of the material at its destination, and Buyer treating the material as outlined in the product data sheet and/or kit insert after arrival. Failure of Buyer to give said notice within said thirty (30) days, or failure of Buyer in treating the material as outlined in the product data sheet and/or kit insert shall constitute a waiver by Buyer of all claims hereunder with respect to said material. The responsibility of all patent considerations in the use of our products rests solely with the user.

Assay Principle

This ELISA utilises specific binding between antibodies and vitellogenin (Vtg) to quantify Vtg in samples from zebrafish (Figure 2; Nilsen et al, 2004). The wells of microplates have been pre-coated with a specific Capture antibody that binds to Vtg in standard and sample added to the wells. A different Vtg-specific Detecting antibody is added to create a sandwich of Vtg and antibody, which is detected with an enzyme-labelled Secondary antibody. The enzyme activity is determined by adding a substrate that gives a coloured product, and the colour intensity is directly proportional to the amount of Vtg present.

Additional Reagents and Equipment Required

In addition to the reagents supplied with the kit, the following reagents and equipment are required and/or recommended to perform the assay:

• 2M H2SO4 (stop solution)
• Microplate reader (wavelength 492 nm)
• Pipettes with disposable tips (5-1000 µl)
• Multi-channel or stepper pipette with disposable tips (50 and 100 µl)
• Test tubes (1-50 ml)
•  Microplate washing device (a manual or automatic plate washer is recommended, but a squeeze bottle or a multichannel/stepper pipette can also be used)
• Vortexer
• Crushed ice

Important Notes

Vtg standard - Vtg is an unstable molecule, and all sample and standard dilutions should be prepared and kept on ice. Reconstituted Vtg can not be frozen and re-used quantitatively at a later date. A dilution series prepared from freshly reconstituted Vtg standard should be run in every assay. A five-plate kit contains two vials of Vtg, sufficient to run two separate assays (for example 2+3 plates). One standard curve is sufficient for a full five-plate assay.

Samples- The assay has been developed for quantification of Vtg in whole body homogenate (wbh), but may also be used with other sample types like plasma. Since compounds in the sample matrix may interfere non-specifically with the assay, usually leading to an underestimation of Vtg at low sample dilutions, the recommended minimum dilution to avoid this matrix effect is 1:500 for wbh (determined for adult zebrafish diluted 1:2 [weight:volume] during sample preparation). For other sample types and sample preparation methods, the minimum dilution factor must be determined in each case.

Techniques.

 In order to obtain reliable results, several common sources of error should be avoided. Important factors to increase reliability are:

• Careful and precise pipetting at every step in the assay. Reverse pipetting of the Dilution buffer is recommended to increase reliability.
• Addition of sample and standard dilutions to the plate in triplicates, instead of duplicates, will increase reliability.
• Avoid shaking and excess foaming when preparing dilutions. Using a vortexer is recommended.