Quantification of FMRP in Human and Mouse Tissues By Capture Immunoassays
Objectives: Current methods for quantitating the levels of FMRP are not very sensitive. Our objective was to improve the methods.
Methods: We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies mAb 6B8 (BioLegend) and R477, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP. The assay readily distinguishes between samples from males with fragile X full mutations and samples from normal males. It also differentiates mosaic from non-mosaic full-mutation male samples. Using mAb 5C2 (BioLegend) and R477 (an inhouse rabbit polyclonal to FMRP), we have also developed a similar immunoassay for the quantification of Fmrp in mouse tissues.
Results: We have employed the assay to screen 2000 newborn dried blood spots (DBS) and present their distribution. We found a high variable expression of FMRP that had an average level 7 folder higher than that of normal adults. We also applied the assay in a retrospective study of 76 newborn DBS that had been stored for an extended period and included full mutation males as well as normal individuals. We were able to correctly identify all 4 known male fragile X positive cases among samples stored up to 47 months. We have used the mouse tissue assay to quantify Fmrp in brainstem, cerebellum, hippocampus, and cortex strains of mouse (C57 BL and FVB) in seven weeks-old animals. The latter assay will allow studying the developmental variation of Fmrp expression in different organs.
Conclusions: Our new assay allows for sensitive and accurate quantitation of FMRP, in human and mouse blood and tissues. It is particularly sensitive for detecting and quantitating newborn blood spots on filter paper, as the drying of blood appears to inactivate proteases that reduce FMRP in stored whole blood samples.