A Seminar on Bioassayprinciples of Bioassay

8/11/2019 A Seminar on Bioassayprinciples of Bioassay

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A SEMINAR ON BIOASSAY:PRINCIPLES OFBIOASSAY

AAFTAB ANWARLUQMAN COLLEGE OF PHARMACY

DEPARTMENT OF PHARMACOLOGYGULBARGA


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Selection of Method:-

Quantitative estimation can be doneby... PHYSICAL

PROPERTY LIKE

COLOUR ,FLOURESCENCE

PHYSICAL

PHYSICALPROPERTY &

CHEMICALREACTION

PHYSIO-CHEMICAL

BIOLOGICALPROPERTY USED TO

ESTIMATE ACTIVITY

BIOLOGICAL


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BIOASSAY:

Estimation of concentration or potency of a

substance by measurement of biological

response i t produces.

i.e. Observation of pharmacological effects on

[1] living tissues, or cells

[2] microorganisms

[3] animals

Also known as

BioStandardization

.


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Bioassay generallyemployed on..

Chemical assay is not available.Quantity of sample too small.

Estimate concentration of active principlein tissue extract.e.g..insulin.Estimate pharmacological activity ofunidentified substance,

Measure drug toxicity,Diagnosis & research.Dose of drug required to producetherapeutic effect.


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If active principle of drug is unknown,

e.g. insulin.

Chemical method not available.Chemical composition not known.Purification for chemical assay not possible.

To ascertain the potency hence served asquantitative part of screening procedure.

Investigate function of endogenous mediators.

Measure drug toxicity & unwanted effects .


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Principles of Bioassay:

1)All bioassays(lab.studies , toxicitystudies , clinical trials)must be

comparative against a standard drug orpreparation.

Way of minimizing error.


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Representative of substance serves as basisfor comparative measurement of activity.

In India maintained & distributed by:

Central Drug Research Laboratory,Kolkata.Central Research Institute,Kasauli.

Standard Preparation: -


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2)Standard & new drug should be asfar as possible identical to each other.

So,dose response curve will have

same slope & parallel.

3)Method of comparison preferably(notessentially) test therapeutic propertyof drug.


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4)Method should estimate as far aspossible the error due to biologicalvariation.


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[1] Quantal Assays [ Direct endpoint ]Elicits an All or None response in differentanimals

E.g.. Digitalis induced cardiac arrest in guinea pigs hypoglycaemic convulsions in mice. Digitalis induced head drop in rabbits Calculation of LD50 in mice or rats

[2] Graded Response Assays [mostly ontissues]

Graded responses to varying dosesUnknown dose response measured on sametissue


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Graded:

Here proportionate increase in responses dueto increase in dose or concentration.

E.g. contraction of smooth muscle forhistamine assay.


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I t can be done by fol lowingmethods..

MATCHING BIOASSAY:-Firstlyresponses of test is taken & is

matched with response of standarddrugs.Done till a closed matching is

observed.Corresponding concentrationcalculated.

Employed for small sample size.


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Concentration response curve ofstandard established.

2-3 responses of testis recorded.Selection such that they lie on

LINEAR PORTION of CRC ofstandard drug.

Precision & reliability is better.

INTERPOLATION:-


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M UL TI PL E POI NT BI OASSAYThree point bioassay:- [2+1 dose assay] two standard & one test responses aretaken down.Fast & convenientProcedure [E.g. Ach bioassay]

Log dose response [LDR] curve plotted with varying conc of std Ach solutionsand given test solutionSelect two std doses s1& s2 [ in 1:2 dose ratio] from linear part of LDR [ Letthe corresponding response be S1, S2]

Choose a test dose t with a response T between S1 & S2Record 4 sets data [Latin square: Randomisation reduces error] as follows

s1 s2 t t s1 s2 s2 t s1 s1 s2 t

Plot mean of S1, S2 and T against dose. Calculate Log Potency ratio [ M ] = [ (T S1) / (S2-S1) ] X log d

[d = dose ratio]


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Four point bioassay:-two standards & twotest responses are recorded.

Two responses of standard should lie onlinear portion of CRC in ratio of 1:2.

Test response is by trial & error method.

Employing statistics responses arerecorded.

Most common method used.


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SIX POINT BIOASSAY:-

Three concentration of standard& test are used.

More time consuming method.


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BRACKETTI NG M ETH OD:-

Used when test sample is too small.

Response of test is bracketed betweentwo responses ( greater & smaller ) ofstandard substances.

Precision & reliability is poor.


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MERITS:

Biological products like toxin,anti toxin,sera can be conveniently assayed.

Measure minute(nano mole & Picomole)quantities of active substances .

Can detect active substance without priorextraction or other treatment.


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DEMERITS:Key problem is variability in response.Large number of animal to be used.Expertise in experimentaldesign,execution of assay & analysis ofdata required.Leads to expensive & time consuming.Time related changes ins sensitivity oftest organ.Tachyphylactic responses of substancebeing assayed.


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CAL CUL ATI ON OF DOSES:-

Expressed in mg/kg.Common route of administrationINTRAPERITONIAL route. as..

Quick onset of action, betterabsorption.Volume of injection in rat-0.5 ml/100g

of body weight, in mice 1ml/100g ofbody weight.Make one ml more then required.


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Barbiturates mostly used.

E.g. Phenobarbital sodium(NEMBUTAL).

Produces anesthesia in dose of 35-45mg/kg.

Give intraperitonialy/intravenous routes

Duration 45-60 min.

Others are chloralose (80-100mg/kg,ip or iv)&urethane (1-1.5g/kg,ip or iv).

L ABORATORY ANAESTH ETI CS


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REFERENCES:

Elements ofpharmacology,Goyal,R.k.

Essentials ofpharmacotherapeutics,Barar,F.S.K.Hand book of experimental

pharmacology,kulkarni.s.k.


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ANY QUESTIONSPLZ.


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A Seminar on Bioassayprinciples of Bioassay