A SEMINAR ON BIOASSAY:PRINCIPLES OF BIOASSAY : A SEMINAR ON BIOASSAY:PRINCIPLES OF BIOASSAY AAFTAB ANWARLUQMAN COLLEGE OF PHARMACYDEPARTMENT OF PHARMACOLOGYGULBARGA Selection of Method:- : Selection of Method:- Quantitative estimation can be done by..
. BIOASSAY: : BIOASSAY: Estimation of concentration or potency of a substance by measurement of biological response it produces.
i.e. Observation of pharmacological effects on
 living tissues, or cells
Also known as BioStandardization. Bioassay generally employed on.. : Bioassay generally employed on.. Chemical assay is not available.
Quantity of sample too small.
Estimate concentration of active principle in tissue extract.e.g..insulin.
Estimate pharmacological activity of unidentified substance,
Measure drug toxicity,
Diagnosis & research.
Dose of drug required to produce therapeutic effect. It is only method under following conditions.. : It is only method under following conditions.. If active principle of drug is unknown,
Chemical method not available.
Chemical composition not known.
Purification for chemical assay not possible.
To ascertain the potency hence served as
quantitative part of screening procedure.
Investigate function of endogenous mediators.
Measure drug toxicity & unwanted effects. Principles of Bioassay: : Principles of Bioassay: 1)All bioassays(lab.studies , toxicity studies , clinical trials)must be comparative against a standard drug or preparation.
Way of minimizing error. Slide 7: Representative of substance serves as basis
for comparative measurement of activity.
In India maintained & distributed by:
Central Drug Research Laboratory,Kolkata.
Central Research Institute,Kasauli. Standard Preparation:- Slide 8: 2)Standard & new drug should be as far as possible identical to each other.
So,dose –response curve will have same slope & parallel.
3)Method of comparison preferably(not essentially) test therapeutic property of drug. Slide 9: 4)Method should estimate as far as possible the error due to biological variation. TYPES OF BIOASSAY: : TYPES OF BIOASSAY:  Quantal Assays [ Direct endpoint ]
Elicits an ‘All or None’ response in different animals
Digitalis induced cardiac arrest in guinea pigs
hypoglycaemic convulsions in mice.
Digitalis induced head drop in rabbits
Calculation of LD50 in mice or rats
 Graded Response Assays [mostly on tissues]
Graded responses to varying doses
Unknown dose response measured on same tissue Graded: : Graded: Here proportionate increase in responses due to increase in dose or concentration.
E.g. contraction of smooth muscle for histamine assay. It can be done by following methods.. : It can be done by following methods.. MATCHING BIOASSAY:-Firstly responses of test is taken & is matched with response of standard drugs.
Done till a closed matching is observed.
Corresponding concentration calculated.
Employed for small sample size. INTERPOLATION:- : Concentration response curve of
2-3 responses of testis recorded.
Selection such that they lie on
LINEAR PORTION of CRC of
Precision & reliability is better. INTERPOLATION:- MULTIPLE POINT BIOASSAY : MULTIPLE POINT BIOASSAY Three point bioassay:- [2+1 dose assay] two standard & one test responses are taken down.
Fast & convenient
Procedure [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 [ in 1:2 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 [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] Slide 15: Four point bioassay:-two standards & two test responses are recorded.
Two responses of standard should lie on linear portion of CRC in ratio of 1:2.
Test response is by trial & error method.
Employing statistics responses are recorded.
Most common method used. SIX POINT BIOASSAY:- : SIX POINT BIOASSAY:- Three concentration of standard & test are used.
More time consuming method. BRACKETTING METHOD:- : BRACKETTING METHOD:- Used when test sample is too small.
Response of test is bracketed between two responses ( greater & smaller ) of standard substances.
Precision & reliability is poor. MERITS: : MERITS: Biological products like toxin,anti toxin,sera can be conveniently assayed.
Measure minute(nano mole & Pico mole)quantities of active substances.
Can detect active substance without prior extraction or other treatment. DEMERITS: : DEMERITS: Key problem is variability in response.
Large number of animal to be used.
Expertise in experimental design,execution of assay & analysis of data required.
Leads to expensive & time consuming.
Time related changes ins sensitivity of test organ.
Tachyphylactic responses of substance being assayed. CALCULATION OF DOSES:- : CALCULATION OF DOSES:- Expressed in mg/kg.
Common route of administration INTRAPERITONIAL route. as..
Quick onset of action, better absorption.
Volume of injection in rat-0.5 ml/100g of body weight, in mice 1ml/100g of body weight.
Make one ml more then required. LABORATORY ANAESTHETICS : 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). LABORATORY ANAESTHETICS REFERENCES: : REFERENCES: Elements of pharmacology,Goyal,R.k.
Essentials of pharmacotherapeutics,Barar,F.S.K.
Hand book of experimental pharmacology,kulkarni.s.k. Slide 23: ANY QUESTIONS PLZ….