Detection of Polypeptide Hormones | Human Body | Biology

Polypeptide hormones are secreted into the blood stream and have endocrine functions in living animals. Polypeptide hormones are synthesized from amino acids according to an mRNA template, which is synthesized from a DNA template inside the cell’s nucleus like other proteins. Polypeptide hormones are secreted from the pituitary gland.

Pituitary gland consists of two lobes:

(a) Anterior lobe or adenohypophysis.

(b) Posterior lobe or neurohypophysis.

The anterior pituitary secretes Prolactin, Thyrotrophin or thyroid stimulating hormone (TSH), Growth hormone, Luteinizing hormone, Follicle-stimulating hormone, Adrenocorticotrophic hormone (ACTH).

The posterior pituitary gland secretes Antidiuretic hormone, (ADH) or vasopressin, Oxytocin (Milk or birth hormone).

A. PRL (Prolactin):

Prolactin (PRL), also known as Luteotropic hormone (LTH) is a peptide hormone discovered by Dr. Henry Friesen and is associated with lactation. Prolactin is secreted by anterior lobe of pituitary gland and falls under hypothalamic control. Prolactin is a single polypeptide chain of 199 amino acids. Structure of prolactin is stabilized by three intra-molecular disulfide bonds.

Prolactin has a molecular weight of about 24 KDa. It is structurally similar to growth hormone and placental lactogen. The dominant form of prolactin is non-glycosylated form that is secreted by the pituitary gland.

Prolactin controls the initiation and maintenance of lactation. It binds to specific cell membrane receptors of target organs (breast, adrenal, ovaries, testes, prostate, kidney and liver). Prolactin increases testesterone production and ovarian steriodogenesis. Excessive secretion of prolactin leads to hyper-prolactinemia which is a relatively common disorder in humans.

Reference Levels of prolactin are:

(a) Women – 2.8-29.2 ng/ml

(b) Men – 2.1-17.7 ng/ml

Immunoassay for Prolactin:

Serum is the preferred specimen but sometimes blood is also used as specimen. Prolactin can be detected by the Sandwich ELISA technique.

1. Sandwich ELISA Assay:

For detection of prolactin by sandwich ELISA technique, initially two or more antibodies are directed against different parts of PRL. One of the antibodies is attached to the solid support (test tube walls/microtitre plates). Another antibody is labeled with a signal molecule (e.g., radioisotope, enzyme or chemiluminescent) and is used as a detector.

Initially radioimmunoassay (RIA) has been used to detect prolactin. But at present, immunometric or sandwich assays are widely used due to the following advantages:

i. They can detect low levels of prolactin.

ii. They are more precisec.

iii. They are more specific.

iv. They have less cross-activity with Growth Hormone.

2. Indirect Approach:

To detect prolactin by indirect approach involves directing antibodies against prolactin and then coupling of this antibody with biotin. Then the prolactin- antibody-biotin complex is separated by avidin linked to a solid phase.

B. Thyroid Stimulating Hormone (TSH):

Thyroid-stimulating hormone or thyrotropin is a peptide hormone secreted by thyrotropic cells of the anterior pituitary gland. TSH regulates the endocrine function of the thyroid gland. TSH stimulates the thyroid gland to secrete the hormones thyroxine (T4) and triiodothyronine (T3). Thyroid-stimulating hormone has a molecular weight of about 26.6 KDa. TSH has two subunits – alpha and beta subunits.

i. Alpha subunit – It consists of 88 amino acids and is common to FSH, LH and hCG.

ii. Beta subunit – It consists of 112 amino acids and is specific to TSH.

A standard range for TSH is 0.4 and 5.0 mlU/L. A high level of TSH (hyperthyroidism) means an underactive thyroid gland is not responding adequately to the stimulation of TSH due to some type of acute or chronic thyroid dysfunction. A low TSH level (hypothyroidism) indicates an overactive thyroid gland. Thyrogen, (drug) manufactured by Genzyme Corp. in Cambridge, Massachusetts is used in patients with thyroid cancer which is related to tumoral factors. A TSH assay is a recommended screening tool for thyroid disease.

Immunoassays for TSH:

i. Enzyme multiplied Immunoassay Technique (EMIT).

ii. Cloned Enzyme Donor Immunoassay (CEDIA).

iii. Equilibrium Dialysis.

iv. Ultrafiltration.

v. Chromatography.

vi. Radioimmunoassay.

vii. Time resolved Fluorescence.

viii. Chemiluminescence (CLEIA).

ix. Mass spectrometry.

C. Growth Hormone (GH):

The identification, purification and synthesis of growth hormone, also known as Somatotrophin is associated with Choh Hao Li. It is the most abundant hormone produced by anterior lobe of pituitary gland. GH has a molecular weight of 21.5 KDa. GH is a single chain polypeptide of 191 amino acids and has 2 intra-molecular disulphide bridges.

GH increases height in children and adolescents, increases calcium retention, and strengthens and increases the mineralization of bone, increases muscle mass through sarcomere hyperplasia, promotes lipolysis, increases protein synthesis, stimulates the growth of all internal organs excluding the brain, role in fuel homeostasis, reduces liver uptake of glucose, promotes gluconeogenesis in the liver, contributes to the maintenance and function of pancreatic islets and stimulates the immune system. High levels of growth hormone indicate – Acromegaly, Gigantism, Growth hormone resistance and pituitary tumor. Low levels of growth hormone indicate – Growth hormone deficiency and hypo-pituitarism.

Immunoassay for Growth Hormone:

1. Competitive Binding Immunoassay:

In Competitive binding Immunoassay, unlabelled GH in test specimen competes with I¹²⁵ labelled GH for a limited amount of primary (Polyclonal) GH antiserum. The complex is then precipitated using a secondary antibody. Radioactivity in the precipitate is measured. The amount of radioactivity is inversely proportional to the amount of unlabelled GH in the sample A.

2. Immuno Radiometric Assays (IRMAs):

In this monoclonal antibodies are used, one of which is attached to solid support and another is radiolabeled with I¹²⁵. Fluoroscent or enzyme labels are also used. These assays are more specific, sensitive and can detect smaller amounts. I-antiGH + Sample (GH) + anti GH*E + [S] → read.

D. Luteinizing Hormone:

Luteinizing Hormone, also known as lutropin, is released by the pituitary gland in response to luteinizing hormone- releasing hormone (LHRH). LH is a hetero-dimeric glycoprotein consisting of one alpha and one beta subunit which makes the functional protein. Alpha and beta subunits are non-covalently associated.

Alpha subunit contains 92 amino acids and beta subunit consists of 121 amino acids. LH structure is similar to follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (hCG). LH levels are normally low during childhood and high after menopause in women. LH controls the length and sequence of the female menstrual cycle, including ovulation, prepares the uterus for implantation of a fertilized egg, and ovarian production of both estrogen and progesterone. In males, LH stimulates the testes to produce and rogen, also known as interstitial-cell-stimulating hormone (ICSH).

Immunoassay for Luteinizing Hormone:

i. Sandwich ELISA.

ii. RIA.

iii. Two site immunometric assay.

E. Follicle-Stimulating Hormone:

FSH is secreted by gonadotropes of the anterior pituitary gland. FSH is a dimeric glycoprotein. Its structure is similar to LH, TSH, and hCG. The protein dimer contains 2 polypeptide units, alpha and beta subunits. The alpha subunits of LH, FSH, TSH, and hCG are identical and contains 92 amino acids. Beta subunit of FSH consists of 118 amino acids. Molecular weight of FSH is 30000. FSH regulates the development, growth, pubertal maturation and reproductive processes of the body. In both males and females, FSH stimulates the maturation of germ cells.

Immunoassay for Follicle-Stimulating Hormone:

i. Chemiluminescence Detection Based on Competitive Immunoassay.

ii. RIA.

iii. Sandwich ELISA.

iv. Two site immunometric assay.

E. Human Chorionic Gonadatrepin (HCG):

Human Chorionic gonadatrepin is a glycoprotein hormone produced by placenta. hCG hormone increases rapidly during pregnancy and reaches 100,000 mIU/ml during first trimester. hCG consists of 244 amino acids and has a molecular mass of 36.7 kDa. It is heterodimeric glcoprotein, with α (alpha) subunit and β (beta) subunit.

The α (alpha) subunit consists of 92 amino acids and the β-subunit consists of 145 amino acids, α (alpha) subunit is identical to Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), Thyroid-Stimulating Hormone (TSH). Human chorionic gonadotropin interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. Human chorionic gonadotropin also plays a role in cellular differentiation/proliferation and may activate apoptosis.

Immunoassay for Human Chorionic Gonadatrepin:

1. Two site sandwich immunoassay.

2. VISIPREG STRIP pregnancy test- It is a rapid, sensitive and accurate antibody based assay for detection of hcG in urine.

Procedure:

Strip containing a dried conjugate of anti hCG is dipped in urine. Urine sample rises up by capillary action and results in reconstitution of the dried conjugate. If hCG is present, it reacts with anti hCG conjugate and forms a complex of anti hcG conjugate-hcG which migrates forward on the membrane pre-dispersed with antiserum anti hCG.

As the antibody of the immobilized anti-hCG is able to capture hCG molecules of the migrating complex, a visible purple band is formed along the exact location of immobilized anti-hCG antiserum. If hCG is absent- anti-hCG conjugate will pass the immobilized anti-hCG band and no line will appear. Above the anti-hCG band, is a control region consisting of a band of immobilized antiserum. This band binds all the conjugates and forms a coloured line regardless of whether hCG is present or absent. Appearance of control line assures correct testing procedures.