Principles, Prerequisites

and Types of assays

Dr. Siddhartha Dutta

MAMC, New Delhi

Outline• Introduction

• Types of assays

• Characteristics of a good assay

• Chemical assays and techniques

• Immunological assays and techniques

• Microbiological assays and techniques

• Bioassay

• Conclusion

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

Types of Assays

• Chemical assays

• Immunoassays

• Microbiological assays

• Bioassay

Characteristics of a good

assay method

• Sensitivity

• Specificity

• Repeatability

• Reproducibility

• Validity

• Stability – tissue has to stay “bioassay-fit

5

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

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

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

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

Spectrophotometry

• Covers 200- 750 nm

• Sophisticated and Sensitive

• Vs. colorimeter- Precisely selected wavelength & manipulated,

monochromator, quartz cuvette

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

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

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

Column chromatography• Adsorption chromatography

• Partition chromatography

• Adv- wide variety of mixture can be separated

• Disad- time, plenty of mobile phase required, expensive

Paper chromatography• Ascending

• Descending

• Rf= dis travelled by compound/dis travelled by solvent

• Adv- simple/rapid/inexpensive

• Disadv- small amount can be tested

Thin layer chromatography

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

High performance liquid chromatography

• Normal phase

• Reverse phase

• Adv- sensitive to very less conc.(ppt), short time, high

resolution better separation, highly reproducible results

Immunoassay

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

Prerequisites

• ANTIGEN

• ANTIBODY

• ANALYTE

• LABEL

Principle

• Immunoassay uses antibody and antigen complexes as a

means of generating measurable result

Types

• Competitive immunoassays.

• Non-competitive immunoassays.

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.

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.

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

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

• 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

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.

Types • ELISA

• Radioimmunoassay

• Fluoroimmunoassay

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)

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.

Types of Elisa

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.

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.

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)

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

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.

• 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

• 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).

• After determining the ratio

of bound to free antigen

in each unknown, the

antigen concentrations

can be read directly from

the standard curve.

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

• 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.

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)

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

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

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

Bioassay

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

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

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

• 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..

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

Types Of Bioassay

Quantal

Graded

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

Graded Bioassay

Effect is produced gradually depending on dose.

E.g. Contraction of smooth muscle preparation

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.

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.

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

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

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

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)

• 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.

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

• 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

Drawbacks

• Biological variation

• Troublesome

• Time consuming

• Expensive

• Less accurate than physico-

chemical methods

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