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.