1. ASSAY IN DRUG ANALYSIS
Dr.Gurumeet C Wadhawa ,
Assistant Professor,
Department of Chemistry.
Rayat Shikshan sansthas Veer Wajekar ASC College,Phunde,Uran
2. Outline
Introduction
Types of assays
Characteristics of a good assay
Chemical assays and techniques
Immunological assays and techniques
Microbiological assays and techniques
Bioassay
Conclusion
3. Assay
An assay is an investigative procedure for
qualitatively assessing or quantitatively measuring
the presence or amount or the functional activity of a
target entity (the analyte) which can be a drug or
biochemical substance or organic sample
5. Characteristics of a good
assay method
Sensitivity
Specificity
Repeatability
Reproducibility
Validity
• Stability – tissue has to stay “bioassay-fit
5
6. Chemical assays
• A chemical assay refers to the analysis of a
sample material, called analyte, using a set of
chemical procedures
Qualitative- extraction, distillation, precipitation and other
methods that determine physicochemical properties
• Quantitative- volume or weight of the substance
8. Photometry
When light is passed through a coloured solution, certain
wavelengths are selectively absorbed giving a plot of the
absorption spectrum of the compound in solution
• The light that is not absorbed is transmitted through the
solution and gives the solution its colour
• Transmittance (T)
• Absorbance (A)= log1/T
• Beer-Lambert law
9. Colorimetry
Colourless compounds are converted into coloured compounds
using chemical reactions under defined reaction conditions
The quantity of colour formed is proportional to the quantity of
the original colourless compound
• Colorimeter
• Pros- inexpensive, quantitative estimation of colored
compounds, easily transportable
• Cons- colorless compounds, UV/IR regions, specific
wavelength
• Uses- protein, glucose estimation in various biochemical
samples
14. Flame photometry
Principle- Matter absorbs light at same wavelength at which it
emits light
Adv-simple/inexpensive/specific/sensitive
to even ppm
15. Chromatography
Differential affinities of the various components of the analyte
towards the stationary and mobile phase results in the
differential separation of the components.
Mobile phase or carrier solvent moving through the column
Stationary phase or
adsorbent
substance that stays fixed inside the
column
Eluent fluid entering the column
Eluate
fluid exiting the column (that is collected
in flasks)
Elution
the process of washing out a compound
through a column using a suitable solvent
Analyte
mixture whose individual components
have to be separated and analyzed
16. Column chromatography
Adsorption chromatography
Partition chromatography
Adv- wide variety of mixture can be separated
Disad- time, plenty of mobile phase required, expensive
19. Gas chromatography
Mobile phase is a gas such as helium and the stationary phase
is a high boiling point liquid adsorbed onto a solid
• Time taken for a particular compound to travel through the
column to the detector is known as its retention time
20. High performance liquid chromatography
Normal phase
Reverse phase
Adv- sensitive to very less conc.(ppt), short time, high
resolution better separation, highly reproducible results
25. An immunoassay is a test that uses antibody and antigen
complexes as a means of generating measurable result
or
An immunoassay is an analytical method which uses
antibodies as reagents to quantitate or detect specific
analytes
29. Competitive Format
In competitive formats, unlabelled analyte (usually antigen)
in the test sample is measured by its ability to compete
with labeled antigen in the immunoassay.
• The unlabeled antigen blocks the ability of the labeled
antigen to bind because that binding site on the antibody is
already occupied.
30. One step competitive format
• Both the labeled antigen reagent (Ag*) and the unlabeled specimen
(or test sample analyte) compete for a limited amount of antibody.
31. Two step competitive
format
• The antibody concentration of the reaction solution is present in
excess in comparison to the concentration of antigen
• Antibody reagent is first incubated with specimen containing
antigens of interest; then in the second step, labeled antigen is
added
• Improved assay sensitivity compared to one step assay formats
32.
33.
34. Noncompetitive (Sandwich) Method
“Sandwich” assay because analyte is bound (sandwiched)
between two highly specific antibody reagents
• Provides highest level of assay sensitivity and specificity
• Applied to the measurement of critical analytes such as cardiac
and hepatitis markers
35.
36. • One step or two step methods
• The two step assay format employs wash steps in which the sandwich
binding complex is isolated and washed to remove excess unbound
labeled reagent and any other interfering substances
37. Homogeneous and Heterogeneous
Immunoassay Methods
• Immunoassay methods that require separation of bound Ab-Ag*
complex are referred to as heterogeneous immunoassays. Those that
do not require separation are referred to as homogeneous
immunoassays.
• Homogeneous methods have been generally applied to the
measurement of small analytes such as abused and therapeutic drugs.
40. ELISA (Enzyme-Linked Immunosorbent
assay)
Enzyme immunoassay
• Both qualitative and quantitative measurement of Ag-Ab
binding
• Direct, Competitive, sandwich ELISA- Ag measurements
• Abs- indirect ELISA
• Advantages (sensitivity, ease of handling multiple samples)
without the disadvantages of dealing with radioactivity (like in
RIA)
41. Prerequisites
• Purified antigen (to detect or quantify antibody).
• Purified antibody (detect or quantify antigen).
• Standard solutions (positive and negative controls).
• Sample to be tested.
• Microtiter dishes: plastic trays with small wells in which the assay is
done.
• Wash fluid (buffer).
• Enzyme-labeled antibody and enzyme substrate.
• ELISA reader (spectrophotometer) for quantitative measurements.
43. Performing the
Tes
t
The tubes are filled with the antigen solution (e.g., urine) to be
assayed. Any antigen molecules present bind to the
immobilized antibody molecules.
• The antibody-enzyme conjugate is added to the reaction
mixture. The antibody part of the conjugate binds to any
antigen molecules that were bound previously, creating an
antibody-antigen-antibody "sandwich".
• After washing away any unbound conjugate, the substrate
solution is added.
• After a set interval, the reaction is stopped (e.g., by adding 1 N
NaOH) and the concentration of colored product formed is
measured in a spectrophotometer. The intensity of color is
proportional to the concentration of bound antigen.
44.
45. Isotopic immunoassay
Based on competition for antibody between radioactive
indicator antigen and unlabelled antigen in test sample.
Increase in count of unlabeled antigen in test sample decrease
the labeled antigen in bound.
The concentration of the test antigen can be determined by
comparison with a standard calibrated curve with known
concentration of purified antigen.
46. Nonisotopic
immunoassay
Differ from isotopic immunoassay in:-
o Type of label used
o Means of end point detection
o Possibility of eliminating a separation test
Two types of nonisotopic immunoassay
are:-
o Fluoroimmunoassay
o ELISA (Enzyme-Linked Immunosorbent assay)
47. Radioimmunoassay
Principle- competitive binding of radiolabelled antigen &
unlabelled antigen to a high affinity antibody
Involves the separation of a protein (from a mixture) using the
specificity of antibody - antigen binding and quantitation using
radioactivity
Adv- faster, higher sensitivity/specificity
Disadv- health hazard, short shelf life, expensive instrument &
needs purified antigen and antibody
48. The
techniqu
e
A mixture is prepared of
o radioactive antigen
Because of the ease with which iodine atoms can be
introduced into tyrosine residues in a protein, the
radioactive isotopes 125I or 131I are often used.
o antibodies against that antigen.
Known amounts of unlabeled ("cold") antigen are added to
samples of the mixture. These compete for the binding sites of
the antibodies.
49. At increasing concentrations of unlabeled antigen, an
increasing amount of radioactive antigen is displaced from
the antibody molecules.
• The antibody-bound antigen is separated from the free
antigen in the supernatant fluid, and the radioactivity of
each is measured
50. The main drawbacks to radioimmunoassay are the expense
and hazards if preparing and handling the radioactive
antigen.
• Both 125I or 131I emit gamma radiation that requires special
counting equipment;
• The body concentrates iodine atoms — radioactive or not —
in the thyroid gland where they are incorporated in thyroxine
(T4).
51.
52. After determining the ratio
of bound to free antigen
in each unknown, the
antigen concentrations
can be read directly from
the standard curve.
53. Fluoroimmunoassay
Fluorescent compound in labeling the antibodies and antigen
• Europium fluoresces when it excite by light in the presence of a
developing solution
• It is 10 times more sensitive than RIA
54. A modern fluorescent based immunoassay uses as the
detection reagent a fluorescent compound which
absorbs light or energy (excitation energy) at a specific
wavelength and then emits light or energy at a different
wavelength
• The difference between the wavelength of the excitation
light and the emission light is called the Stokes shift.
55. Application of immunoassay
in food Industry
Many of the macromolecule that can found in food are
good antigen and antibodies are capable of recognizing
them and small molecule.
• Composition of raw material and final product
• Harmful and useful minor substances with biological
activity (toxins and allergens)
• Enzyme detection
• Contaminants detection and determination (hormones
,drug, pesticides residue)
56. Microbiological assays
Principle- Based upon a comparison of the inhibition of
growth of micro-organisms by measured concentration of the
antibiotics to be examined with that produced by known
concentrations of a standard preparation of the antibiotic having
a known activity
1. The cylinder-plate
(or cup-plate) method
2. The turbidimetric
(or tube assay) method
60. Definition
Comparative assessment of relative potency of a test compound to
a standard compound on a living or biological tissue
Quantitative measurement of the amount of active principle or
substance in a pharmaceutical preparation or biological material using
a suitable biological system
61. Comparison Of Chemical
& Bioassay
Bioassay
Less Precise
More time consuming
Active constituent &
structure not known.
More sensitive
Chemical Assay
More Precise
Less time consuming
Active constituent &
structure fully established.
Less sensitive
62. Indications Of Bioassay
Chemical method is either
Not available
If available, too complex,
Insensitive to low doses e.g. Histamine
If active principle of drug is not known e.g. insulin
Tomeasure the pharmacological activity of new or
chemically undefined substances
Chemicals with similar structure, but different
biological activity
63. Chemical structure known; cannot be actively purified. Eg:
Peptide hormone
• Active principle cannot be isolated e.g. posterior pituitary
extract, insulin
• To compare the strength of a drug obtained from various
sources due to different compositions (Eg:Cardiac
glycosides,catecholamines)
• Biological activity of drug cannot defined by a chemical assay
e.g. Cis and Trans form of methyl phenidate.
• For biological standardization of drugs obtained from natural
sources as these cannot be obtained in pure form. Eg:
Oxytocin, Vasopressin, Insulin, Heparin..
64. Principles of bioassay
To compare the test substance with the International Standard
preparation of the same
• To find out how much test substance is required to produce
the same biological effect, as produced by the standard
• Activity assayed should be the activity of interest
• Standard & test sample - similar pharmacological effects &
mode of action
62
65. both should be compared for their established
pharmacological effect using specified technique
Ex: *Ach – contractile response on frog rectus
*Histamine – contractile response on guinea pig ileum
Problem of biological variation must be minimized
Experimental conditions - kept constant
Animals - same species, sex and weight
Number of animals - large enough to minimize error (individual
variation)
Isolated preparations - sensitive
63
66. Prerequisites for Bioassay
Physiological salt solutions
Kymograph: Sherrington- starling kymograph
Student Organ bath
lever
68. Quantal
All or none response in all individuals,
e.g. Digitalis induced cardiac arrest in guinea pigs
hypoglycemic convulsions in mice by insulin and
Calculation of LD50 in mice or rats
Not précise
Employed for:
• Comparison of LD50 and ED50
69. Graded Bioassay
Effect is produced gradually depending on dose.
E.g. Contraction of smooth muscle preparation
70. Accuracy Limits Of Bioassay
“Accuracy improves the efficiency of bioassay for
pharmaceutical biological products.”
An accuracy within ± 20 % of true value is good.
An accuracy within ± 10 %
of truevalue is excellent.
71. Various Physiological salt
solutions
For 10 litres
pH- 7.3-7.4
Frog-
Ringer
Kreb’s Tyrode Ringer-
Locke
De
Jalon
Mc
Ewen
NaCl 65 g 69 g 80 g 91.5 g 90 g 76 g
KCl 1.4 g 3.5 g 2.0 g 4.2 g 4.2 g 4.2 g
MgCl². 6H²O --- 1.1 g 1.0 g --- --- ---
NaH2PO4.
H²O
0.1 g 1.4 g 0.5 g --- --- 1.4 g
NaHCO³ 2 g 21 g 10 g 1.5 g 5 g 21 g
CaCl² 1.2 g 2.8 g 2 g 2.4 g 0.6 g 2.4 g
Glucose 20 g. 20 g. 10 g. 10 g. 5 g. 20 g
Aerating
Gas
air O² +
5%CO²
O² or
air
Pure O² O² + 5%
CO²
O² + 5%
CO²
•Calcium chloride to be added last.
•Calcium chloride and magnesium chloride are hygroscopic, so use stock
72. Uses: Physiological salt Solutions
Physiological salt
solutions
Uses
Frog-Ringer Amphibian tissue preparation
Kreb’s Mammalian/Avian skeletal
muscle preparation
Tyrode Intestine preparation
Ringer-Locke Heart muscle preparation
De Jalon Rat uterus preparation
74. Step 2: Arrange the instrument
and adjust the water bath.
Kymograph: Sherrington-
starling kymograph
To obtain a graphical amplified measurable
response of a muscle or tissue
Two important parts: motor box and drum
Speed lever: 1 revolution/ 96 min.
Paper:
glossy side outside – least resistance
Rough side inside – stick to the drum.
Fixing solution: shellac and colophony
saturated in alcohol
75. Student Organ bath
Outer bath:-
First designed by rudolph
magnus
Perpex glass
Store water outside the inner
bath to maintain the
temperature
Inner bath:-
o Glass
o To observe the tissue during
experiment
o 5-50ml (usually 10ml)
76. Tissue holder and oxygen supply:-
Tissue is attached inside the inner water bath to a tissue holder.
Also supports the oxygen supply to the tissue.
77. Step:3 -Balance the lever
Lever:
Three basic parts:
Effort arm- where force in applied
Load arm- where effect of force is
observed
Fulcrum
Classes of lever – 3
Types of lever
78. Magnification :
= Distance from the fulcrum to the writing point
Distance form the fulcrum to the tied tissue
o For slow contracting muscles:- 10-15 times
o For fast contracting muscles:-5-10 times
