Bioassay

BIOASSAY

·         Bio-living, assay-test

·         It is the process of determining the potency of the drug by using suitable biological system like animals, tissues, microbes etc.

PRINCIPLE OF BIOASSAY

·         To compare the test substance with the International Standard preparation (IP, BP, USP) of the same.

·         To find out how much test substance is required to produce the same biological effect, as produced by the standard.

APPLICATION

·         To determine the potency of a drug.

·         To determine the dose of a drug required to produced a therapeutic or toxic response (ED50, LD50).

·         This process is used in the development of new molecules or preparation.

CHARACTERISTICS OF A GOOD ASSAY METHOD

·         Sensitivity

·         Specificity

·         Repeatability

·         Reproducibility

·         Precision

·         Accuracy

·         Stability – tissue has to stay “bioassay-fit

BIOASSAY CAN BE PERFORMED ON

1.       In-vivo Intact animal

2.       In-vitro isolated tissues, specific cells, organisms.

·         WHOLE ANIMALS

o    Nor Adrenaline – Spinal Cat

o    Cardiac Glycosides – Guinea Pig

o    Insulin – Mice

o    Estrogens – Ovariectamised Female Rat

·         MICRO ORGANISMS

o    Vit B12 – Euglena gracilis

o    Tetracycline – Bacillus pumilus

·         ISOLATED TISSUE

o    Acetyl Choline – Frog Rectus Abdominus muscle

o    Histamine – Guinea Pig ileum

o    Adrenaline – Rat uterus

o    Oxytocin – Rat uterus oestrogen primed

·         DISPERSED CELLS

o    Plasma LH estimation by stimulation of testosterone synthesis – on isolated Leydig cells

 

TYPES OF BIOASSAY

·         There are mainly two types of bioassay;

1.       Quantal assay

§  Direct end point assay (DEPA)

§  LD50 determination

2.       Graded assay

§  Matching

§  Bracketing

§  Interpolation

§  Multiple point

    Three point

    Four point

    Six point

 

§  Cumulative dose response 

QUANTAL ASSAY

·         The response is in the form of “all or none”, i.e. either no response or maximum response. Drugs producing quantal effect can be bioassayed by end point method.

·         The threshold dose producing a predetermined effect is measured

·         Comparison between the results of standard and the test

 

·         E.g: Bioassay of digitalis in cats, Insulin induced hypoglycemic convulsions in rat.

 

GRADED ASSAY

 

·         Response is proportional to the dose and response may lie between no response and the maximum response.

MATCHING OR BRACKETING

·         A constant dose of the standard is bracketed by varying dose of test sample.

·         until an exact matching between the response of standard & that of the test sample is matched.

 

·         Strength of unknown/test drug can be found by simple interpolation of bracketed response.

ADVANTAGES

·         Simple & Faster method.

·         Amount of test drug available is small

·         Does not involve complicated calculations

·         Does not depend on DRC

DISADVANTAGES

·         less accurate

·         Time consuming

·         Cannot get exact match of response

·         Quantitative difference between test & standard not obtained.

INTERPOLATION ASSAY

·         The concentration of test drug is interpolated form the dose response curve graph (DRC).

·         The DRC of standard drug is plotted first, then the different concentrations of the test drug are plotted.

 

·         The dose of the test drug which comes at the linear log dose-response relationship is interpolated from the dose response plot.

MULTIPLE POINT ASSAY

·         The responses are taken several times and mean of each is taken. So that the chances of errors are minimized.

·         These assays are performed by the selection of 1 or more dose responses of test compound and these responses are compared with 2 or more responses of standards.

THREE-POINT BIOASSAY

·         In this bioassay total three responses selected from DRC for standard as well as test.

·         One for test and two standard responses are selected from DRC.

·         This method depends on the latin square randomization of three responses from DRC.

·         The response selection is made between 25-75%.

 

·         Estimates of relative potency are then obtained as the displacement of parallel log dose response lines of standard and test compound.

Latin square randomization

FOUR-POINT BIOASSAY

 

·         Four-point bioassay implies 2 responses of standard and 2 of test from the DRC for the consecutive 16 response of Latin square randomization.

Latin square randomization

SIX-POINT BIOASSAY

·         The responses obtained for the 6-point is ‘36’ and ‘64’ for 8-point.

·         Less adopted because of time consuming, but it reduces error.

CUMULATIVE DOSE RESPONSE CURVE

·         Increase concentration of drug in bath fluid step by step without washing out the preceding doses.

·         Continue till super-maximal effect is seen.

 

·         Dose response curve is plotted.

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