A concise study material on assays, types of assay, principle of assays, prerequisites of various assays

1. Principles,
Prerequisites
and
Types of assays
Dr. Siddhartha Dutta
MAMC, New Delhi

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

4. Types of Assays
• Chemical assays
• Immunoassays
• Microbiological assays
• Bioassay

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

7. Physicochemical assay techniques
1. Photometry
2. Colorimetry
3. Spectrophotometry
4. Fluorimetry
5. Flame photometry
6. Chromatography
7. Column chromatography
8. Paper chromatography
9. Thin layer chromatography
10.Gas chromatography
11.High performance liquid chromatography

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

12. Spectrophotometry
• Covers 200- 750 nm
• Sophisticated and Sensitive
• Vs. colorimeter- Precisely selected wavelength & manipulated,
monochromator, quartz cuvette

13. Fluorimetry
• Principle
• Intensity of fluorescence ∝ concentration
• Specific and sensitive
• Pharmaceutical analysis
• Adrenaline
• Cyanocobalamin
• Riboflavin
• Morphine
• Pentobarbitone
Drugs Excitation
wavelength
Emission
wavelength
Hydrocortisone 460 nm 520 nm
Nicotinamide 250 nm 430 nm

14. Flame photometry
• Principle- Matter absorbs light at same wavelength at which it
emits light
Adv-simple/inexpensive/specific/sensitive
to even ppm
Dis-conc cant be measured/high conc-
wrong result

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

17. Paper chromatography
• Ascending
• Descending
• Rf= dis travelled by compound/dis travelled by solvent
• Adv- simple/rapid/inexpensive
• Disadv- small amount can be tested

18. Thin layer chromatography

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

21. Immunoassay

22.  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

23. Prerequisites
• ANTIGEN
• ANTIBODY
• ANALYTE
• LABEL

24. Principle
• Immunoassay uses antibody and antigen complexes as a
means of generating measurable result

25. Types
• Competitive immunoassays.
• Non-competitive immunoassays.

26. 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.

27. 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.

28. 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

31. 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

33. • 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

34. 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.

36. Types
• ELISA
• Radioimmunoassay
• Fluoroimmunoassay

37. 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)

38. 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.

39. Types of Elisa

40. Performing the Test
• 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.

42. 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.

43. 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)

44. 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

45. The technique
• 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.

46. • 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

47. • 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).

49. • After determining the ratio
of bound to free antigen
in each unknown, the
antigen concentrations
can be read directly from
the standard curve.

50. 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

51. • 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.

52. 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)

53. 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

54. Antibiotic Test Organism ATCC1 No.
Amikacin
Amphotericin B
Bacitracin
Bleomycin
Carbenicillin
Chlortetracycline
Erythromycin
Framycetin
Gentamicin
Kanamycin sulphate
Neomycin
Novobiocin
Nystatin
Oxytetracycline
Polymyxin B
Spiramycin
Streptomycin
Tetracycline
Staphylococcus aureus
Saccharomyces cerevisiae
Micrococcus luteus
Mycobacterium smegmatis
Pseudomonas aeruginosa
Bacillus pumilus
Micrococcus luteus
Bacillus pumilus
Bacillus subtilis
Staphylococcus epidermidis
Bacillus pumilus
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus epidermidis
Saccharomyces cerevisiae
Bacillus cereus var, mycoides
Staphylococcus aureus
Bordetella bronchiseptica
Bacillus pumilus
Bacillus subtilis
Klebsiella pnumoniae
Bacillus cereus
Staphylococcus aureus
29737
9763
10240
607
25619
14884
9341
14884
6633
12228
14884
29737
12228
12228
2601
11778
29737
4617
6633
6633
10031
11778
29737

55. Buffers
Buffer No.
Dipotassium
Hydrogen
Phosphate,
K2HPO4
Potassium
Dihydrogen
phosphate,
KH2PO4
pH adjusted
after
sterilization to
1
2
3
4
5
6
2.0
16.73
-
20.0
35.0
13.6
8.0
0.523
13.61
80.00
-
4.0
6.0±0.1
8.0±0.1
4.5±0.1
6.0±0.1
10.5±0.1*
7.0±0.2

56. Bioassay

57. 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

58. 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

59. 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
• To measure the pharmacological activity of new or
chemically undefined substances
• Chemicals with similar structure, but different biological
activity

60. • 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..

61. 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

62. • 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

63. Prerequisites for Bioassay
• Physiological salt solutions
• Kymograph: Sherrington- starling kymograph
• Student Organ bath
• lever

64. Types Of Bioassay
Quantal
Graded

65. 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

66. Graded Bioassay
 Effect is produced gradually depending on dose.
 E.g. Contraction of smooth muscle preparation

67. 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 true value is
excellent.

68. Various Physiological salt solutions
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
For 10 litres
pH- 7.3-7.4
•Calcium chloride to be added last.
•Calcium chloride and magnesium chloride are hygroscopic, so use stock

69. 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

70. Electrolytes
Ingredients Functions
NaCl Maintain osmolarity
K+ Nerve conduction, muscle
contraction, maintain heart rate &
rhythm
Ca + Contraction
Mg+ Neurotransmission , decrease
spontaneous activity
NaHCO³ &
NaH2PO4
Buffer
Glucose Nutrient

71. 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

72. 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)

73. • 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.

74. 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

75. • 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

76. Drawbacks
• Biological variation
• Troublesome
• Time consuming
• Expensive
• Less accurate than physico-
chemical methods
77

77. THANK YOU