Validating the Use of ImageJ for the QuantitativeAnalysis of Immunofluorescence

We sought to validate the use of the free software ImageJ to calculate weighted luminescence (WL) as an outcome measure for immunofluorescence. The noise signal seen in direct immunofluorescence (DIF) demonstrates an inverse relationship with the concentration of added antibody. Thus, the drop in this antigen-independent signal which accounts for the majority of each image allows for mathematical modelling of signal intensity. DIF was performed on specimens from 10 bullous pemphigoid (BP) patients and 6 pemphigus vulgaris (PV) patients. DIF was prepared using dilutions of tagged antibodies for IgG and C3 at a dilution ranging from 1:100 to 1:800. Images were processed using ImageJ, with green luminescence histograms generated for each dilution. To determine the relationship of mean WL as the dilution of antibody increased, we generated histograms with logarithmic regression curves for anti-IgG and anti-C3 antibodies. To account for the role of antigendependent signal (i.e. the immunoreactive portion of the DIF picture), an additional regression curve limited to dilutions ranging from 1:100 to 1:400 was performed to reduce the risk of the Fc receptor or C3 becoming unsaturated. Logarithmic regression for BP IgG and C3 dilutions demonstrated a significant and moderate to strong correlation, (R2=0.51, P<0.001) and (R2=0.50, P<0.001) respectively with a mild drop in relationship strength, but still statistically significant when corrected for antigen-dependent signal. A weaker relationship was seen in PV, but this also was statistically significant regardless of correction. WL is potentially a valuable measure for quantitative comparison of fluorescent signaling in the experimental setting. Further uses of comparative immunofluorescence using ImageJ such as comparing antibody binding or antigen expression require validation of WL which we herein demonstrate.