ANALYTICAL TECHNIQUES IN PHARMACOLOGY –

1) ELISA –

ANSWER:

- It stands for Enzyme-Linked Immunosorbent Assay, an immunoassay

- USES :
o It is a qualitative assay for detecting antigen of interest.
o A type of ELISA called sandwich ELISA is a semi quantitative method (can
detect as well as measure a molecule).
o It can also be used to detect the presence of an antibody.
o It can also be used to measure an unknown concentration of a molecule
from a standard curve, formed from a known concentration of antigen
or antibody.
- ADVANTAGES :
o It is a semi-quantitative method of detecting a molecule
o It is quicker in detecting an unknown
o It is a versatile technique
o It is highly specific and sensitive
- DISADVANTAGES :
o It is a costly procedure
o It is dependent on the precision of method used by each investigator –
subject to bias
o It is not completely quantitative
o It does not yield instantaneous results
- PARTS :
o ENZYMES COATED ON A PLATE (STRIP / PLATE ELISA) – e.g. ALP, Horse
Radish peroxidase (HRP) & galactosidase.
o Flat bottom plates / strips
o Coating buffer
o Wash buffer
o Blocking buffer
o Test samples
o Primary antibodies
o Secondary antibody conjugates
o Substrate
o Reaction stop solution
o Micropipettes
o Micro tips
- PRINCIPLE :

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 o For detecting an antigen, corresponding antibodies are immobilized
onto a solid medium / support
o Test injected into this support
o Incubated
o Secondary antibody to a different epitope allowed to react, after getting
conjugated with the enzyme
o Incubated and washed to remove unreacted compounds
o Changes in colour is indicative of required compound
- TYPES –
o Dot ELISA
o PLATE / STRIP ELISA

2) HPLC –

- It stands for High-Performance Liquid Chromatography.

- It also stands for High Pressure Liquid Chromatography / High Resolution
Chromatography.
- USES –
o It is a technique used to separate substances in a mixture (liquid).
o It is used in pharmacological assays e.g. in Micro dialysis techniques
used in assessing many drugs in neuropsychopharmacology (S.K.Gupta)
- PRINCIPLE –
o It is based on adsorption, partition, exclusion and ion exchange
o Any one of above four / a combination of these can be done based on:
particle size & polarity of substances to be separated.
- TYPES –
o Reverse phase partition chromatography (RPPC)
o Ion-exchange chromatography (IEC)
o Exclusion chromatography (EC)
o Adsorption chromatography (AC)
o Normal phase partition chromatography (NPPC)
- PARTS –
o Pump – to deliver pulse less low flow rate, high pressure solvent; 2
types: constant pressure & constant flow pumps
o A sampling valve to inject the sample
o Steel (stainless to avoid attack from dissolved gas in the mixture) / glass
columns – to move the stationary phase into mobile phase
o Column packings for adsorption, ion exchange, partition (normal &
reverse phase) of mixtures – made usually of silica

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 o Detectors to detect each component using UV / Fluorescence – types
are: general & specific
o Derivatives to detect specific chemical groups – e.g. –COOH group to
detect coumarin derivatives using fluorescence
- ADVANTAGES –
o Choosing a good mobile phase (a solvent) has more separative capacity
than in GLC
o Speedy separation
o Sensitive
o High resolution
o Separations within 15 – 20 minutes
o Easy recovery of pure analytes possible
o Columns can be reused
o Less initial sample preparation needed
- DISADVANTAGES –
o BIG INVESTMENT NEEDED / Costly
o Technical skill needed for maintenance

3) LIQUID CHROMATOGRAPHY –

- Write about HPLC plus –

- Liquid Chromatography is of many types:
- Liquid-liquid partition chromatography
- HPLC
- It is used to separate a liquid mixture in a liquid solvent.
- ADVANTAGES –
o Low cost
o Easy application of analytical mixture

4) GAS CHROMATOGRAPHY –

- Here Nitrogen is the mobile inert gaseous phase, which passes through a column
containing a stationary phase (a mixture to be separated).
- PRINCIPLE –
o After reacting with the stationery phase, the gas phase goes to a
detector that detects the substance based on a unique retention time.
- TYPES – Depending on the state of stationery phase, 2 types:
o GSC = Gas-solid chromatography
o GLC = Gas-liquid chromatography

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 - PARTS OF GLC –
o Support media – Teflon (e.g.)
o Stationery phase – a liquid
o Columns – types: packed, capillary; are inert to hold the stationery phase
in the outer tube of a concentric pipe – the inner hole is for the nitrogen
to pass through
o Carrier gases – nitrogen
o Katharometer / infra red spectrophotometric detector / electron capture
detector / etc. for detection of chromatographic peaks using retention
times
- PROCEDURE –
o HIGH TEMPERATURE  Sample introduction into a column in a
solvent  mobile gas phase  derivative formation 
detection
- DISADVANTAGES C / T HPLC –
o Less separation capability
o Cannot separate large / thermolabile / non volatile molecules
easily.
o Nitrogen must be at very low temperatures
o Columns cannot be re used.

5) SPECTROPHOTOMETER / MASS SPECTROMETRY –

- Beer Lambert ‘s law states that –

o Conc. of unknown / conc. of std = absorbance of unknown / absorbance of
std
- Spectrophotometry, is a type of mass spectrometry, used to detect absorbance
of unknown and standard quantitatively, using a colorimeter
- PARTS –
o Light source – UV, Visible / near IR spectrum
o Wavelength filters – absorption & interference filters to select
wavelengths entering the colorimeter
o Optical systems & slits associated with it
o Sample holders / cells / cuvettes
o Micropipettes to load the sample
o Single beam / double beam optical paths
o Photosensitive detectors – barrier layer cells, photoconductive /
photoemittive / photomultiplier cells
o Output devices

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 - TYPES –
- Single beam
- Double beam spectrophotometers
- BLANKS ARE USED TO ADJUST THE INSTRUMENT BEFORE THE TEST SAMPLE IS
LOADED
- USES –
o To find out the concentration of an unknown
o In many Pharmacogenomic experiments as a part of AUTOANALYZER
APPARATUS
o In many animal & in vitro assays used in drug discovery
o Used in toxicology

6) Radioimmunoassay (RIA) –

o Is a quantitative method to detect compounds in serum using:

o TYPES:
o Radioactive labeled antibodies(IMMUNORADIOMETRIC ASSAY /
IRMA) or
o Enzymes (ENZYME IMMUNOASSAY / EIA), detected using:
o Fluorescence (Fluoroimmunoassay / FIA) OR
o Luminescence (LUMINESCENCE IMMUNOASSAY / LIA).
o It was also called as displacement / saturation analysis
o ADVANTAGES –
o Sensitive
o Specific
o Accurate
o DISADVANTAGES –
o High dilution reagents  prolonged reaction time
o Gamma emitting radioisotope  short shelf life & hazardous
o Add reagent very precisely as it is highly sensitive
o Limited assay range
o Lack of linearity between signal & concentration
o Difficulty in automation
o Lengthy counting time
o PRINCIPLE –
o It uses a radioactive labeled antibody tagged to an antigen in an
antiserum
o Amount of antibody to be used can be obtained from
equilibrium constant Ka of antibody with antigen

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 oAmount of labeled antigen to be added to antibody at each
dilution is plotted against logarithm of standard concentration
o EXAMPLE OF ITS USE –
o Used in studying any new drug acting on thyroid gland

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REFERENCES:
1) VARLEY’S PRACTICAL CLINICAL BIOCHEISTRY 6/E ALAN H GOWENLOCK
CBS 2002.
2) A BASIC MANUAL OF PRACTICAL IMMUNOTECHNIQUES 1/E
SOLOMON PAUL JP?

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