BIOASSAY TECHNIQUES Submitted to

Dr. Sujarani S.

Asst. Prof.

Dept. of VPT, Pookode.

Submitted by

Dr. Sindhu K.

MVSc scholar,

Dept. of VPT, Pookode.

Biological assessment.

• Estimation or determination of concentration or potency of a physical, chemical or biological substance (agent) by means of measuring and comparing the magnitude of the response of the test with that of standard over a suitable biological system under standard set of conditions.

• The estimation of the concentration or potency of a substance by measurement of the biological response that it produces.

• The structure of bioassay: STIMULUS-applied to subject.

RESPONSE-of the subject to the stimulus.

The purpose of bioassay.

• To ascertain the potency of a drug and hence it serves as the quantitative part of any screening procedure (Research).

• To standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptics etc., so that each contains the uniform specified pharmacological activity. (standardization required as these are all used over biological system in some or other form.)

• Helps to determine the specificity of a compound to be used e.g. Penicillin's are effective against Gram +ve. but not on Gram –ve.

• From the clinical point of view, bioassay may help in the diagnosis of various conditions. e.g. gonadotrophins for pregnancy.

• Sometimes the chemical composition of samples are different but have same biological activity.

• Certain complex compounds like Vitamin B-12 which can't be analysed by simple assay techniques can be effectively estimated by Bioassays.

• For samples where no other methods of assays are available.

Principle of Bioassay.

To compare the test substance with the International Standard preparation ofthe same and to find out how much test substance is required to produce thesame biological effect, as produced by the standard.

The standards are internationally accepted samples of drugs maintained andrecommended by the Expert Committee of the Biological Standardization ofW.H.O.They represent the fixed units of activity (definite weight of preparation) for drugs.

In India

• standard drugs are maintainedin Government institutions like

1. Central Drug ResearchInstitute, Lucknow

2. Central Drug Laboratory,Calcutta.

Classification of bioassay.

•Invitro.

•Invivo.

•Exvivo.

In vitro techniques:

• These techniques employ a cell culture of recommended biological system to study the effect of compound under standard condition not similar to that of living environment. Here the cell culture survives by utilization of the nutrition in the media.

• Ex: use of stem cells,

cell culture,

microbes (bacteria) etc.

In vivo techniques:

• These techniques employ a livinganimal recommended for thepurpose of assay. The techniquesaims to study the biological effector response of the compound underscreening in a living system directly.

• Ex: By use of rodents, rabbits etc.

Ex vivo techniques:

• These techniques employ a tissue or cells ofrecommended living system to study theeffect of compound under test in suitableconditions within the stipulated time oforgan survival outside the body.

• Ex: Use of any isolated organ from animalsin a glass ware to study the effect ofcompound within the period of its survivaloutside the living body with provision ofonly oxygen, glucose and isotonic salts tomaintain cell & cell organelles integrity.

Types of bioassay.

• Qualitative bioassayis used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity.

Eg: Arnold Adolph Berthold's famous experiment on castrated chickens. This analysis found that by removing the testes of a chicken, it would not develop into a rooster because the endocrine signals necessary for this process were not available.

• Quantitative bioassays involve estimation of concentration/potency of a substance by measurement of the biological response it produces. These bioassays are typically analyzed using the methods of biostatistics.

Bioassay Methods.

1. Graded Response Assay: : In these assays, as the dose increases there is an equivalent rise in response. The potency is estimated by comparing the Test sample responses with the standard response curve.

• Conc. of unknown= Threshold dose of standard/threshold dose of test x Conc. of standard.

• E.g. Acetyl-choline producing contraction in the muscle of frog Rectus abdominis.

2. End Point or Quantal Assay: As the name indicates, the threshold dose of the sample required to elicit a complete or a particular pharmacological effect is determined and compared with standard.

• E.g., Digitalis producing cardiac arrest.

• Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is done by this method.

Based on the method used during the grade point assay procedure for determination of Type of activity and Potency of the Sample, four methods of assays are classified as:

• Matching point or bracketing method

• Interpolation assay

• Three point (2+1) assay

• Four- point (2+2) assay

Matching point or bracketing method:

• Here a constant dose of the standard is bracketed by varying dose of sample until an exact matching between the standard dose responses and the particular dose response of the sample is achieved.

This technique is used

• when test sample is too small

• Inaccurate & margin of error

difficult to estimate

Eg: histamine on guinea pig ileum,

Posterior pituitary on rat uterus.

Interpolation assay.

• Bioassays are conducted by determining the amount ofpreparation of unknown potency required to produce adefinite effect on suitable test animals/organs/Tissueunder standard conditions.

• This effect is compared with that of a standard. Thusthe amount of the test substance required to producethe same biological effect as a given quantity the unitof a standard preparation is compared and the potencyof the unknown is expressed as a % of that of thestandard by employing a simple formula.

Multi point Bioassay.

• This method incorporates the principleof interpolation and bracketing.

• 2+1 indicates- Two response of Standardand one response of Test respectively.

• This procedure of 2+1 or 2+2 is repeated3 times or 4 times based on the methodwith crossing over of all the samples.

• It can further divided as 3 point, 4 pointand 6 point bioassay.

Three point assay [2+1 dose assay]

• Fast & convenient:

• Log dose response [LDR] curve plotted with varying conc of std drug solutions and given test solution

• Select two std doses s1& s2 [ in 2:3 dose ratio] from linear part of LDR [ Let the corresponding response be S1, S2]

• Choose a test dose t with a response T between S1 & S2

• Record 4 sets data as follows

• s1 s2 t

• t s1 s2

• s2 t s1

• s1 s2 t

• Log Potency ratio [M] = [(T –S1) / (S2-S1)] X log (dose ratio)

4 point assay [2 +2 dose assay]

• [E.g. Ach bioassay]

• Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution

• Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response be S1, S2]

• Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ;

• Also s2/s1 = t2/t1 = 2/3

Record 4 data sets

• s1 s2 t1 t2

• s2 t1 t2 s1

• t1 t2 s1 s2

• t2 s1 s2 t1

Other bioassay’s.

• Immunological assay (ELISA).

• Micro-bioassay.

• Radioimmunoassay.

• Biotechnology.

ELISA (immunological assay)

• ELISA is a popular format of a "wet-lab"type analytic biochemistry assay that usesa solid-phase enzyme immunoassay (EIA)to detect the presence of a substance,usually an antigen, in a liquid sample orwet sample.

• The ELISA has been used as a diagnostictool in medicine and plant pathology, aswell as a quality-control check in variousindustries.

• The substances detected by ELISA testsinclude hormones, bacterial antigens andantibodies.

Types of ELISA.• Direct ELISA: involve attachment of the antigen to the solid phase, followed

by an enzyme-labeled antibody. This type of assay generally makesmeasurement of crude samples difficult, since contaminating proteinscompete for plastic binding sites.

• Indirect ELISA: involve attachment of the antigen to a solid phase, but in thiscase, the primary antibody is not labeled. An enzyme-conjugated secondaryantibody, directed at the first antibody, is then added. This format is used mostoften to detect specific antibodies in sera.

• Competitive ELISA: involves the simultaneous addition of 'competing'antibodies or proteins. The decrease in signal of samples where the secondantibody or protein is added gives a highly specific result.

• Sandwich ELISA: involve attachment of a capture antibody to a solid phasesupport. Samples containing known or unknown antigen are then added in amatrix or buffer that will minimize attachment to the solid phase. An enzyme-labeled antibody is then added for detection.

Applications of ELISA

• ELISA Test Applications in Antibody Concentration Determination

• ELISA Test Applications in Monoclonal Antibody Screening

• ELISA Test Applications in Virus test (HIV, West Nile Virus, NDV)

• ELISA Test Applications in Home Pregnancy Test

• ELISA Test Applications in Food industry (detecting potential food allergens such as milk, peanuts, walnuts, almonds and eggs)

• ELISA Test can be used to diagnostic diseases

Micro-bioassay (antibiotics)

• The potency (activity) of an antibiotic product is expressed as the ratio of the dose that inhibits the growth of a suitable susceptible microorganism to the dose of an International Biological Standard, an International Biological Reference Preparation, or an International Chemical Reference Substance of that antibiotic that produces similar inhibition.

• To carry out the assay a comparison is made between the inhibition of the growth of microorganisms produced by known concentrations of the reference material and that produced by measured dilutions of the test substance.

• This response can be measured by the diffusion method or in a liquid medium by the turbidimetric method.

Radioimmunoassay:

• RIA is a very sensitive in vitro assay technique used to measure concentrations of antigens (E.g. hormone levels in the blood) by use of antibodies.

• It is the estimation of the concentration of the substance in a unit quantity of preparation using radiolabelled antigens.

• It requires special precautions and licensing, since radioactive substances are used.

• A number of drugs are estimated now days by radioimmunoassay methods because these methods are highly specific and highly sensitive.

• Eg: the radioimmunoassay of insulin is based on the ability of human insulin (unlabelled) to displace beef’s insulin (which may be labelled) from the binding sites (i.e. antibodies).

Principle of radioimmunoassay.

It uses an immune reaction [antigen-antibody reaction] to estimate a ligand.

Ag+Ag*+Ab → [Ag -Ab+ Ag*Ab + Ag + Ab*]

• - Unbound Ag* and Ag washed out

• - Radio activity of bound residue measured.

• - Ligand concentration is inversely related to the radio activity.

• - [Ag: ligand to be measured; Ag*: radiolabelled ligand].

Method of RIA.• Requirements:

• 1. Preparation and characterization of an antigen

• 2. Radio labeling of the antigen

• 3. Preparation of the specific antibody

• 4. Development of assay system.

• Components of RIA Assay Kit:

• Drug

• Antibody

• Labeled Drug

Applications of RIA.Endocrinology

• Insulin, HCG, Vasopressin

• Detects Endocrine Disorders

• Physiology of Endocrine Function

Pharmacology

• Morphine

• Detect Drug Abuse or Drug Poisoning

• Study Drug Kinetics

Epidemiology

• Hepatitis B

Clinical Immunology

• Antibodies for Inhalant Allergens

• Allergy Diagnosis

Oncology

• Carcino embryonic Antigen

• Early Cancer Detection and Diagnosis.

Biotechnology.

• “Any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use" (UN Convention on Biological Diversity, Art. 2)

• The American Chemical Society defines biotechnology as the application of biological organisms, systems, or processes by various industries to learning about the science of life and the improvement of the value of materials and organisms such as pharmaceuticals, crops, and livestock.

• The European Federation of Biotechnology defines Biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

• Biotechnology also writes on the pure biological sciences (animal cell culture, biochemistry, cell biology, embryology, genetics, microbiology, and molecular biology).

Branches of biotechnology.

• Bioinformatics

• Blue biotechnology

•Green biotechnology

• Red biotechnology

•White biotechnology

Bioinformatics

• Is an interdisciplinary field whichaddresses biological problems usingcomputational techniques, and makes therapid organization as well as analysis ofbiological data possible.

• It plays a key role in various areas, suchas functional genomics, structuralgenomics, and proteomics, and forms akey component in the biotechnology andpharmaceutical sector.

Blue biotechnology

• is a term that has been used to describe the marine and aquatic applications of biotechnology.

Green biotechnology

• is biotechnology applied toagricultural processes.

• Eg:

1. The selection and domestication ofplants via micropropagation.

2. The designing of transgenic plantsto grow under specific environments inthe presence (or absence) of chemicals

Red biotechnology

• is applied to medical processes.

• Eg:

1.The designing of organisms to produce antibiotics,

2.The engineering of genetic cures through genetic manipulation.

White biotechnology

• Also known as industrial biotechnology,

is biotechnology applied to industrial processes.

• Eg:

1. The designing of an organism to produce auseful chemical.

2. The using of enzymes as industrial catalyststo either produce valuable chemicals or destroyhazardous/polluting chemicals.

White biotechnology tends to consume less inresources than traditional processes used toproduce industrial goods.

Applications

• Health care (medical),

• Crop production and agriculture,

• Non food (industrial) uses of crops

and other products

(e.g. biodegradable plastics, vegetable oil, biofuels),

• Environmental uses.

To Sum - up

• Bioassay & its principles, structure.

• Types & methods of bioassay.

• Immunological assay (ELISA).

• Micro-bioassay.

• Radioimmunoassay.

• Biotechnology.

Thank-you