Intended use

This immunoassay kit allows for the specific measurement of Human Follistatin (FS) concentrations in cell culture supernates, serum, and plasma.

 

Introduction

Follistatin (FS) was first identified as a follicle-stimulating hormone inhibiting substance

present in ovarian follicular fluid . It has since been shown to be a multifunctional regulatory protein exerting a majority of its effects via neutralization of activin. FS is a monomeric binding protein that regulates activin activity by forming an inactive complex. FS plays a role as a tissue regulator in the gonad, pituitary gland,pregnancy membranes, vasculature, and liver. It is also essential for normal development, as FS-knockout mice die shortly after birth with a range of defects including insufficient muscle development and skeletal abnormalities.

FS is a single-chain polypeptide with a structure unlike activin or inhibin. Alternative splicing

events, proteolytic cleavage and variable glycosylation lead to a vast array of possible FS

isoforms found within various biological fluids. FS is a member of a larger group of proteins that contain a well-conserved secondary structure that has been termed the “follistatin” domain. This domain consists of a cysteine-rich sequence with similarity to EGF and the Kazal family of enzyme inhibitors. FS family members have an insertion in the ovomucoid-like inhibitory loop that prevents protease activity within the

FS domain . FS is commonly found co-localized within a tissue with activin subunits or activin receptors. Activin/FS complexes may also bind extracellular matrix components thus forming reservoirs of activin/FS. Within the circulation,70 - 90% of FS exists in the bound state .

FS binds to the common _A and _B subunits of activin and inhibin. Therefore, activin has two

binding sites for FS (i.e. one activin dimer and two FS monomers make up the activin-FS

complex), whereas inhibin has only one . Activin A, AB, or B each binds FS with similar

affinity . Since inhibin contains only one _-subunit, it binds FS with a lower affinity. It is currently unknown whether or not FS binds additional inhibin subunits.

 

Test principle

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for FS has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any FS present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for FS is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of FS bound in the initial step. The color development is stopped and the intensity of the color is measured.

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