assays, types of assays, principle and prerequisites of assays and bioassay
TRANSCRIPT
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Principles, Prerequisites
and Types of assays
Dr. Siddhartha Dutta
MAMC, New Delhi
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Outline• Introduction
• Types of assays
• Characteristics of a good assay
• Chemical assays and techniques
• Immunological assays and techniques
• Microbiological assays and techniques
• Bioassay
• Conclusion
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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
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Types of Assays
• Chemical assays
• Immunoassays
• Microbiological assays
• Bioassay
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Characteristics of a good
assay method
• Sensitivity
• Specificity
• Repeatability
• Reproducibility
• Validity
• Stability – tissue has to stay “bioassay-fit
5
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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
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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
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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
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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
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Spectrophotometry
• Covers 200- 750 nm
• Sophisticated and Sensitive
• Vs. colorimeter- Precisely selected wavelength & manipulated,
monochromator, quartz cuvette
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Fluorimetry
• Principle
• Intensity of fluorescence ∝ concentration
• Specific and sensitive
• Pharmaceutical analysis
• Adrenaline
• Cyanocobalamin
• Riboflavin
• Morphine
• Pentobarbitone
Drugs Excitation wavelength
Emissionwavelength
Hydrocortisone 460 nm 520 nm
Nicotinamide 250 nm 430 nm
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Flame photometry• Principle- Matter absorbs light at same wavelength at which it
emits light
Adv-simple/inexpensive/specific/sensitive to even ppmDis-conc cant be measured/high conc-wrong result
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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
Eluatefluid exiting the column (that is collected in flasks)
Elutionthe process of washing out a compound through a column using a suitable solvent
Analytemixture whose individual components have to be separated and analyzed
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Column chromatography• Adsorption chromatography
• Partition chromatography
• Adv- wide variety of mixture can be separated
• Disad- time, plenty of mobile phase required, expensive
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Paper chromatography• Ascending
• Descending
• Rf= dis travelled by compound/dis travelled by solvent
• Adv- simple/rapid/inexpensive
• Disadv- small amount can be tested
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Thin layer chromatography
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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
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High performance liquid chromatography
• Normal phase
• Reverse phase
• Adv- sensitive to very less conc.(ppt), short time, high
resolution better separation, highly reproducible results
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Immunoassay
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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
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Prerequisites
• ANTIGEN
• ANTIBODY
• ANALYTE
• LABEL
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Principle
• Immunoassay uses antibody and antigen complexes as a
means of generating measurable result
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Types
• Competitive immunoassays.
• Non-competitive immunoassays.
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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.
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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.
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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
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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
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• 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
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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.
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Types • ELISA
• Radioimmunoassay
• Fluoroimmunoassay
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ELISA (Enzyme-Linked Immunosorbent
assay)
• Enzyme immunoassay
• Both qualitative and quantitative measurement of Ag-Abbinding
• 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)
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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.
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Types of Elisa
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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.
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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.
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Nonisotopicimmunoassay
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)
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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
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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.
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• 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
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• 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).
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• After determining the ratio
of bound to free antigen
in each unknown, the
antigen concentrations
can be read directly from
the standard curve.
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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
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• 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.
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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)
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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
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Antibiotic Test Organism ATCC1 No.
AmikacinAmphotericin B
BacitracinBleomycin
CarbenicillinChlortetracycline
ErythromycinFramycetin
GentamicinKanamycin sulphate
NeomycinNovobiocin
NystatinOxytetracycline
Polymyxin BSpiramycin
Streptomycin
Tetracycline
Tobramycin
Staphylococcus aureusSaccharomyces cerevisiae
Micrococcus luteusMycobacterium smegmatisPseudomonas aeruginosa
Bacillus pumilusMicrococcus luteus
Bacillus pumilusBacillus subtilis
Staphylococcus epidermidisBacillus pumilus
Staphylococcus aureusStaphylococcus epidermidisStaphylococcus epidermidis
Saccharomyces cerevisiaeBacillus cereus var, mycoides
Staphylococcus aureusBordetella bronchiseptica
Bacillus pumilusBacillus subtilis
Klebsiella pnumoniaeBacillus cereus
Staphylococcus aureusStaphylococcus aureus
29737976310240607
2561914884934114884663312228148842973712228122282601117782973746176633663310031117782973729737
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Buffers
Buffer No.
DipotassiumHydrogen
Phosphate,K2HPO4
PotassiumDihydrogenphosphate,
KH2PO4
pH adjusted after
sterilization to
123456
2.016.73
-20.035.013.6
8.00.52313.6180.00
-4.0
6.0±0.18.0±0.14.5±0.16.0±0.1
10.5±0.1*
7.0±0.2
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Bioassay
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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
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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
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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 orchemically undefined substances
• Chemicals with similar structure, but different biologicalactivity
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• 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..
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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
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• 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
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Prerequisites for Bioassay
• Physiological salt solutions
• Kymograph: Sherrington- starling kymograph
• Student Organ bath
• lever
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Types Of Bioassay
Quantal
Graded
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QuantalAll 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
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Graded Bioassay
Effect is produced gradually depending on dose.
E.g. Contraction of smooth muscle preparation
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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.
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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 solution.
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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
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Electrolytes Ingredients Functions
NaCl Maintain osmolarity
K+ Nerve conduction, musclecontraction, maintain heart rate & rhythm
Ca + Contraction
Mg+ Neurotransmission , decrease
spontaneous activity
NaHCO³ & NaH2PO4
Buffer
Glucose Nutrient
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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
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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)
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• 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.
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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
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• 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
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Drawbacks
• Biological variation
• Troublesome
• Time consuming
• Expensive
• Less accurate than physico-
chemical methods
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THANK YOU