Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #4120 Procedure for Enzyme Immunoassay (EIA) of Salmon/Trout IGF-II

For a detailed procedure on how to perform an EIA see Protocol
#3020 General Procedure for Microplate Enzyme Immunoassay (EIA)

The Salmon/Trout IGF-II enzyme-immunoassay (EIA) was carried out according to this method with the following modifications.

This assay was first published and its performance reported in Wilkinson et al. (2004).

Reagents
Salmon/Trout IGF-II (Receptor Grade)
Product Code: ATU020 20 µg
Product Code: ATU100 100 µg

IGF-II, Anti-Salmon/Trout
Product Code: PAAA1 100 µl

Reconstitute the vial in 100 µl sterile distilled or Milli-Q water. Allow to stand at room temperature for at least 10 minutes before use.

Plate coating
Coat wells with 100 µl of a 50 ng / ml recombinant Salmon/Trout IGF-II solution in carbonate coating buffer (0.015 M Na₂CO₃, 0.035 M NaHCO₃, pH 9.6). Plates are then incubated for 16 hours at 4 °C.

Washing and Blocking of plates
Following two consecutive washes with 300 µl PBS-T (PBS, 0.05 % Tween-20) block the wells by incubating for 1 hour at room temperature with 200 µl PBS, 2 % BSA (bovine serum albumin). Between all subsequent steps in the EIA, five consecutive washes were performed as described in Protocol #3020 above.

Acid treatment and pre-incubation of standards and samples
Recombinant Salmon/Trout IGF-II standards (1,000 to 0.46 ng/ml) are prepared by serial dilution in EIA dilution buffer (PBS-T, 0.1 % BSA). Assay standards and plasma samples are acid-treated to release IGF-II from IGFBP complexes based on methods adapted from Diamandi et al. (1998). Briefly, incubate 100 µl standard or plasma samples for 30 minutes at room temparature with 70 µl 0.1 M glycine.HCl (pH 2.0). The acidified mix is then neutralized with 30 µl 0.2 M Tris (pH 10). Following acid-treatment the standard and plasma samples (200 µl total volume) are incubated for 1 hour (complex time) at room temperature (shaking) with 200 µl IGF-II, Anti-Salmon/Trout used at a working dilution of 1:1000 in EIA dilution buffer.

Following pre-incubation of the standards and plasma samples with IGF-II, Anti-Salmon/Trout, 3 x 100 µl aliquots of each are removed and transferred to the blocked EIA plate, and incubated for 30 minutes (binding time) at room tempaerature (with gentle shaking).

Signal amplification with Biotin-anti-rabbit Ig and Streptavidin-HRP
After washing the plates as described above, signal amplification is performed by first adding 100 µl, donkey biotin-anti-rabbit Ig (Amersham Biosciences, UK. Ltd.) at 1:4000 working dilution in EIA dilution buffer for 1 hour at room temperature with shaking. Streptavidin-HRP (100 µl) (R&D Systems, Minneapolis, MN) was subsequently added (after another wash step) at 1:200 working dilution in EIA dilution buffer and incubated for 1 hour at room temparture with shaking.

Enzymatic colour reaction
Following another wash step as described above a colour reaction was performed on the plates by adding 100 µl substrate buffer (40 mM citric acid, 67 mM Na₂HPO₄, pH 5.0) containing 0.1 % H₂O₂ (30 % [w/w] stock solution) and 5 mM O-Phenylenediamine (OPD). Following 5-minute colour development the reaction was terminated via the addition of 50 µl 2 M H₂SO₄. Absorbance was subsequently read at 492 nm using a Labsystems (Multiskan EX) microplate reader.

References:
Diamandi, A., Khosravi, M.J., Mistry, J., Martinez, V., Guevara-Aguirre, J. (1998) Filter paper blood spot assay of human insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 and preliminary application in the evaluation of growth homone status. J. Clin. Endo. Met. 83, 2296 – 2301.

Wilkinson R. J., Elliott, P., Hohmann, A., Francis, G., Carragher, J. (2004) Development and characterization of a competitive polyclonal antibody enzyme-immunoassay for salmon insulin-like growth factor-II. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 139, 193 - 201.