BY: Reddy Sailaja M (MSIWM030)

HORMONES

A hormone is a signaling molecule secreted by endocrine glands in response to physiological stimuli in multi cellular organisms. These hormones circulate in blood and reach its destination to exert specific function. Hormones help maintain physiological and behavioral functions in the organisms.

Hormones are classified into three main classes:

  • Steriod hormones – Lipid soluble and move across plasma membrane of the targeted cells.
  • Peptide hormones – Water soluble and act through cell surface receptors present on the targeted cells.
  • Aminoacid derivatives

Table 1: Three classes of hormones

Peptide hormones

Peptide hormones are made up of small amino acid chains called, peptides. Peptide hormones are synthesized in the cells from amino  acids based on mRNA sequence that is derived from DNA template within the nucleus.

Peptide hormones can’t navigate across plasma membrane of the cell. Hence, they exert their function by binding to the receptor present on the cell surface of the target cell that in turn trigger signal transduction and cellular response. Some peptide hormones like parathyroid hormone-related protein, angiotensin II etc interact with intracellular components within cytoplasm or nucleus by an intracrine mode of interaction.

Some of the examples of peptide hormones are as follows:

  • Adrenocorticotrophic hormone
  • Thyroid stimulating hormone
  • Vasopressin
  • Angiotensin II
  • Antrial natriuretic peptide
  • Calcitonin
  • Follicle stimulating hormone
  • Insulin
  • Growth hormone
  • Parathyroid hormone
  • Prolactin

Table 2: Main peptide hormones – details and functions

Detection of peptide hormones

As peptide hormones circulate in blood to reach their destination, serum generally acts as the source of detection and measurement.

The following are the main detection methods used to detection peptide hormones:

  1. Sandwich ELISA technique:
  2. In sandwich ELISA method, two antibodies are used to detect hormone of interest. One of the antibodies is attached to the solid support on the micro titer plate, called as capture antibody.
  3. Second antibody labeled with a signal molecule (enzyme or radioisotope or chemilumiscent) acts as detector.
  4. When the analyte containing mixture is loaded onto the micro titer plate, capture antibodies bind to the analyte via epitope that is present on the analyte surface and catch hold of analytes.
  5. When the detector antibodies are added to the plate, they bind to different location on the analyte.
  6. In the enzyme based reaction, when substrate is added, it reacts with enzyme that is attached to the detector body and shows response in the form of color change.

Figure 1: Sandwich ELISA to detect analytes in the blood

  • Radioimmunoassay (RIA):
  • RIA is an in vitro detection technique that detects and measures antigens (like hormones and other foreign substances) in the blood. RIA technique is discovered by Berson and Yalow in 1960 to analyze insulin levels in blood.
  • RIA method is based on the radioactivity measurement associated with antigen-antibody interactions in the reaction.
  • A known antigen that is radiolabelled is incubated with antibody at known concentrations.
  • When the analyte containing solution is added to the labelled antigen-antibody mixture, antigen of interest replaces labelled antigen and bind to the antibody.
  • More the antigen of interest present in the solution, more labelled antigen will be displaced and replaced with antigen of interest.

Figure 2: Radioimmunoassay

  • Enzyme multiplied Immunoassay Technique (EMIT):
  • EMIT is more easy and equivalent detection method, both qualitatively and quantitatively to measure wide-range of analytes from the serum.
  • EMIT is based on the principle that the amount of analyte present in the solution is directly proportional to the inhibition of enzyme-substrate reaction complex.
  • In this technique, initially a known analyte is labelled with an enzyme and antibody specific to drug is allowed to bind drug-enzyme complex. This results in inhibition of enzyme activity.
  • When the solution containing analyte is added to the above mixture, the analyte releases the antibody from the drug-enzyme complex, thereby increasing enzymatic activity.
  • Therefore, enzyme activity is proportional to the analyte present in the sample added and is measured by absorbance value changes of the enzyme.

Figure 3: Enzyme multiplied Immunoassay Technique

  • Immunoradiometric assay (IRMA)
  • IRMA utilizes radiolabeled antibodies to detect analytes of interest.
  • In this technique, antibody is directly labeled with radioisotopes rather than using two antibodies as in other immune assays.
  • When an analyte containing solution is added to micro titer plateradiolabeled antibodies bind to the specific epitopes of the anlytes and forms the antigen-antibody complex.
  • I125 and I131 radioisotopes used in general for this assay.
  • Unbound radiolabeled antibodies are removed from the plate by second wash during the process.

Figure 4: Immunoradiometric assay

The other peptide hormone detection methods are listed below:

  • Ultrafiltration
  • Chromatography
  • Time resolved fluorescence
  • Mass spectrometry
  • Two site immunometric technique

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