Follicle Stimulating hormone (FSH) is a glycoprotein consisting of two subunits with an approximate molecular mass of 35,500 daltons. The alpha-subunit is similar to other pituitary hormones [luteinizing stimulating hormone (LH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (hCG)] while the beta-subunit is unique.

The beta-subunit confers the biological activity to the molecule. Stimulation by gonadotropin-releasing hormone (GnRH) causes release of FSH, as well as LH, from the pituitary and is transported by the blood to their sites of action, the testes or ovary.

In men, FSH acts on the Sertoli cells of the testis, stimulating the synthesis of inhibin, which appears to specifically inhibit further FSH secretion, and androgen-binding protein. Thus, it indirectly supports spermatogenesis.

In women, FSH acts on the granulosa cells of the ovary, stimulating steroidogensis. All ovulatory menstrual cycles have a characteristic pattern of FSH, as well as LH, secretion. The menstrual cycle is divided into a follicular phase and a luteal phase by the midcycle surge of the gonadotropins (LH and FSH). As the follicular phase progresses, FSH concentration decreases. Near the time ovulation occur, about midcycle, FSH peaks (lesser in magnitude than LH) to its highest level. The clinical usefulness of the measurement of Follicle Stimulating hormone (FSH) in ascertaining the homeostasis of fertility regulation via the hypothalamic - pituitary - gonadal axis has been well established.

FSH ELISA:

Immunoenzymatic colorimetric method (ELISA) for quantitative determination of FSH in serum.

Principle of the Assay:

In this method, calibrators, patient specimens and/or controls (containing the native FSH antigen) are first added to streptavidin coated wells. Biotinylated monoclonal and horseradish peroxidase (HRP) labelled antibodies are added and the reactants are mixed. The different types of antibodies used have high affinity and specificity and are directed against distinct and different epitopes of FSH. Reaction between the various FSH antibodies and native FSH occurs in the microwells without competition or steric hindrance, forming a soluble sandwich complex. The interaction is illustrated by the following equation:

BtnAb(m) = Biotinylated Monoclonal Antibody (Excess Quantity)
AgFSH = Native Antigen (Variable Quantity)
HRP-Ab(p) = HRP labelled Antibody (Excess Quantity)
EnzAb(p)-AgFSH-BtnAb(m)= Antigen-Antibodies Sandwich Complex
ka = Rate Constant of Associationk-a = Rate Constant of Dissociation

Simultaneously, the complex is fixed to the well through the high affinity reaction of streptavidin and biotinylated antibody. This interaction is illustrated below:

Streptavidin C.W. = Streptavidin immobolized on well.
Immobilized complex = sandwich complex bound to the solid surface.

After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by aspiration. The native antigen concentration is directly proportional to the HRP activity in the antibody-bound fraction. The activity of the conjugated HRP is quantified by reaction with TMB substrate to produce blue colour. The reaction is terminated by adding stop solution which turns the blue colour into yellow. The absorbance is measured on a plate reader.

Specific performance characteristics:

Intra Assay Variation:

Within-run precision was determined by replicate determinations of three different control sera in one assay. The within-assay variability is shown below:

Inter Assay Variation:

Between-run precision was determined by replicate measurements of three different control sera in several different assays. The between-assay variability is shown below:

Analytical Sensitivity:

The lowest detectable concentration of FSH by this assay is 0.6 mIU/ml.

Accuracy:

The NovaTec FSH ELISA was compared to a well-established RIA method FSH MAIAclone Adaltis giving the following results:
r = 0.99
n = 162

Order information: