Fish Vitellogenin (VTG) ELISA Kit

This assay employs the competitive enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with goat-anti-rabbit antibody. Standards or samples are added to the appropriate microtiter plate wells with an antibody specific for VTG and Horseradish Peroxidase (HRP) conjugated VTG. The competitive inhibition reaction is launched between with HRP labeled VTG and unlabeled VTG with the antibody. A substrate solution is added to the wells and the color develops in opposite to the amount of VTG in the sample. The color development is stopped and the intensity of the color is measured.

Vitellogenin (VTG or less popularly known as VG) (from latin vitellus, yolk, and gener, to produce) is a precursor protein of egg yolk normally in the blood or hemolymph only of females that is used as a biomarker in vertebrates of exposure to environmental estrogens which stimulate elevated levels in males as well as females. Vitellogenin is a synonymous term for the gene and the expressed protein (citation needed). The protein product is classified as a glycolipoprotein, having properties of a sugar, fat and protein. It belongs to a family of several lipid transport proteins.

Vitellogenin is an egg yolk precursor protein expressed in the females of nearly all oviparous species including fish, amphibians, reptiles, birds, most invertebrates, and monotremes. Vitellogenin is the precursor of the lipoproteins and phosphoproteins that make up most of the protein content of yolk. In the presence of estrogenic endocrine disruptive chemicals (EDCs), male fish can express the Vg gene in a dose dependent manner. Vg gene expression in male fish can be used as a molecular marker of exposure to estrogenic EDCs.

This assay has high sensitivity and excellent specificity for detection of Fish VTG. No significant cross-reactivity or interference between Fish VTG and analogues was observed.

Matrices listed below were spiked with certain level of recombinant Fish VTG and the recovery rates were calculated by comparing the measured value to the expected amount of Fish VTG in samples.

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Fish VTG and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.