Is there an ultrasensitive Perch ELISA available? Are there any suggestions for modifying the Perch ELISA if lower VTG values need to be detected in mucus?
The TECO® Perch Vitellogenin ELISA is the only commercially available ELISA for Perch/Perciformes. At the moment, there is not enough demand for an ultra-sensitive protocol. One option using the current protocol is to lower the dilution factor from 1:10 (50μL sample + 450μL extraction buffer) to 1:6 (50μL sample + 250μL extraction buffer). In development, this has been shown to increase the sensitivity of the assay appropriately.
How do I know mucus samples are not cross-contaminated by a nearby fish when I am collecting my samples?
It is recommended that mucus under the gill be swabbed, if cross contamination from nearby fish is a concern. This can be a concern with net fishing, where high- and low- level vitellogenin fish are in close proximity to each other.
How do I know mucus samples are not cross-contaminated with other environmental substances that may interfere with the assay and affect my results?
The antibodies used in the assays are very specific and do not recognize other components. Furthermore, the extraction buffer (500μL) used to dilute the mucus collected with the swab has a dilution effect that will dilute out a rare occurrence of an environmental contaminant. In development of the assay, aquarium water was used as a negative control and no contamination from the mucus samples was detected in comparison to the aquarium water.
While this is not necessary, the extraction buffer used in collecting mucus and the dilution buffer used in diluting blood and WBH are protein-free so they do allow for an easy parallel determination of proteins.
The calibrator material is biologic and not recombinant. It is prepared from serum that has been run through a column and separated via affinity chromatography. It is extensively validated and characterized using immunohistochemical staining, Western blot and SDS PAGE technology.
The exact composition is proprietary information. The solution is responsible for activating the full reactivity of the plates.
The exact composition is proprietary information but both are protein-free solutions optimized for the assay.
A polyclonal (affinity purified) antibody to vitellogenin.
The best sample dilution will vary based on type of sample (blood, homogenate or mucus), species and exposure to test compound(s). Refer to the package insert and/or the Vitellogenin ELISA Crossreactivity chart for suggested dilution ranges. During assay evaluation/validation, it is recommended that several dilutions, based on the recommended range, be tested initially using a subgroup of samples. The results from assaying the subgroup at several dilutions can then be used to determine the best dilution for the rest of your samples based on your experimental design.
Yes, in all assays. Homogenate epidermal mucus samples may also be used.
Molecular testing reflects the gene expression whereas ELISA assays detect the product, or actual effect, of this expression. Additionally, molecular assays tend to be time-consuming, costly and more sensitive to technique variables such that they require special laboratories with well-trained personnel.
How soon after exposure will the VTG protein be detected in mucus compared to blood/homogenate and compared to the detection of vtg gene expression?
This will vary depending on the substance and species, but in general, the VTG protein (gene expression) can be detected in mucus 48 hours after exposure to 2.5 ng/L EE2.
Mucus collection is non-invasive and non-destructive. It can be easier to collect from smaller fish. Collectingmucus does not require sacrificing the fish which means repeat sampling allows for studying induction kineticsby individual. Repeat sampling also allows for pre-exposure testing, making it possible to assess handling(stress)-related impacts on VTG induction. Additionally, research indicates the vitellogenin protein may be morestable in mucus. In blood, the vitellogenin protein can be especially prone to proteolytic cleavage.