Dosing protocol - ELISA technique
An ELISA kit was used (R&D system Quantikine ELISA) for a quantitative analysis of the total MMP-2, MMP-7 and MMP-9 (free and complex forms) and human TIMP-2 (plate of 96 wells pre-coated with the primary antibody). Conditioning was performed using a saline / EDTA buffer in order to obtain viscosity of the samples to be compatible with this technique. Samples were diluted in a buffer with 0.1 M NaCl, 1mM PMSF, 1x LAP, 5mM EDTA, 20mM Tris 0.6M. Thorough homogenization of the effusions was achieved using a pestle, to manually extract the proteins. The samples were then placed on a circular stirrer for 1 hour at 4°C. A total protein assay was performed using BCA kits (BCA protein assay kit, ThermoFisher Scientific®) on each buffered sample.
We used 1/10 and 1/20 dilutions of the middle ear effusions for the analysis of MMP-2, TIMP-2 and MMP-7 concentrations, and 1/40 for the determination of MMP-9. On each plate, 64 ELISA wells were used to test the duplicate samples. The remaining 16 wells were used to carry out the standard curve with standard solutions.
Analysis was performed by spectrophotometry at 450 nm and 540 nm. The optical density (OD) was the average of the two ODs obtained by each duplicated sample on the microplate reader. A concentration in ng/mL was obtained (ratio of OD to standard curve) ), then this level was normalized by the total protein level of the sample, the final concentration being expressed in ng/µg protein.
Results are presented as an average concentration and standard deviation. The statistical data were calculated using a non parametric test (Mann-Whitney test for independent data).
Ethical and regulatory aspects
This project has been validated by the Local Ethics Committee (IRB of Montpellier) in MR-004. An explanatory note was given to the parents and children included in the study, and consent was collected at the end of the consultation.