Animal DIY ELISA Kits

Yes, there is a technical guide detailing all the reagents, ingredients and protocols to successfully use the Assay Genie DIY ELISA.

The DIY ELISA kits contain validated antibody pairs and highly purified protein standard.

The datasheets for the individual components presents an example of the optimal concentrations to use and will help to determine the number of assays that can be performed with each set.

For example, the Porcine IFNα1 DIY ELISA Kit includes:

100 µg of Anti-Swine IFNα1 Polyclonal Antibody:
The recommended titer for this reagent in ELISA is 1-5 µg/ml. If coating with 100 µl per well in a 96-well plate, this will require approximately 10 ml of reagent to coat a 96-well plate. Thus, 10 µg-50 µg per plate is required, depending on the titer used. This means the reagent provided should coat 2-10 plates.

5 µg of Recombinant Porcine IFNα1:
The optimal standard concentrations will need to be determined by the end-user. If using a top standard concentration of 50 ng/ml (0.05 µg/ml), assuming 0.5 ml of standard is required per plate, this should make enough standard for 200 assays.

50 µg of Biotinylated Anti-Porcine IFNα1 Polyclonal Antibody:
The recommended titer for this reagent in ELISA is 0.1-0.5 µg/ml. If detecting with 100 µl per well in a 96-well plate, this will require approximately 10 ml of reagent to detect a 96-well plate. Thus, 1-5 µg per ELISA is required, depending on titer used. That means the reagent provided should detect in 10-50 assays.

Thus, depending on optimization results, there can be quite a large difference in the number of ELISA performed with the provided components. Each of the components can be purchased individually especially the capture antibody as this tends to be depleted before the other components.

The DIY ELISA can be used with serum, plasma, or other sample types for detection. For other sample types, validation of the Standard and Sample Diluent in the assay is required. The optimal diluent should provide linear results (i.e. when the sample is diluted, similar values are obtained when correcting for the dilution factor) and have 80-120% recovery (i.e. if a known quantity of standard is added to a sample type, 80-120% of what was added should be measured in the assay).

To determine if the sample diluent works with a specific sample type, a linearity and recovery assay should be performed. Simply, prepare a standard curve using your test sample diluent. Add or “spike” a known quantity of standard into the test sample. Add or “spike” the same quantity of standard to the test sample diluent (this is the 100% recovery value). Be sure to run a non-standard spiked test sample in the assay to subtract out any background values from the test sample. Prepare multiple serial dilutions of the diluent standard-spiked sample, the test standard-spiked sample, and the non-standard spiked test sample with the test diluent (i.e. 1:2, 1:4, 1:8, 1:16, 1:32, etc.). Optimal recovery is when the standard spiked sample value (subtract out non-standard spiked sample background) is 80-120% of the diluent standard spiked sample result. In addition, the results should give similar values when corrected for dilution (linearity).

A 1:4 to 1:8 dilution with 4% BSA in DPBS (Reagent Diluent) is sufficient to achieve linearity of the sample and obtain optimal recovery. If optimal results are not obtained with 4% BSA in DPBS, a higher sample dilution or a different sample diluent will need to be used. Other diluents to try are 10-50% serum (such as fetal bovine serum or equine serum) in DPBS (as long as the serum does not contain the protein of interest that could cross-react with the ELISA). Additionally, increasing the BSA content in DPBS buffer may also increase linearity and recovery.