BIOASSAY Submitted to

Dr. Monika DwivediAsst. Prof.

Dept. of Pharmaceutical science

Submitted byGovind yadav

2

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.

3

The purpose of bioassay.• To ascertain the potency of a drug • To standardize drugs, vaccines, toxins or poisons, disinfectants,

antiseptics etc., so that each contains the uniform specified pharmacological activity. • 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.

4

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

5

In India

• standard drugs are maintained in Government institutions like

1.Central Drug Research Institute, Lucknow

2.Central Drug Laboratory, Calcutta.

Bioassay can be performed on

6

Invivo

•Intact animals

Invitro

•Isolated tissues•Specific cells•Organisms

7

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.

8

In vivo techniques:• These techniques employ a

living animal recommended for the purpose of assay. The techniques aims to study the biological effect or response of the compound under screening in a living system directly. • Ex: By use of mice, rabbits,

dog etc.

9

Partial List Of Official Quantitative Biological Tests

Drug and Dosage form Test Animal (s)

Antibiotics Suitable microorganism

Insulin Rabbit & Mice

Digitalis & other glycosides Pigeon & Guinea Pig

Vitamin A & D Rat

Parathyroid drugs Dog

Posterior pituitary Rat

Tubocurarine Chloride Rabbit

Characteristics of a good assay method

• Sensitivity• Specificity• Repeatability• Reproducibility• Precision• Accuracy• Stability – tissue has to stay

“bioassay-fit 10

11

Types of Bioassays

[1] Quantal Assays [ Direct endpoint ]• Elicits an ‘All or None’ response in different animals• Eg. • Digitalis induced cardiac arrest in guinea pigs• hypoglycemic convulsions in mice. • Digitalis induced head drop in rabbits• Calculation of LD50 in mice or rats

[2] Graded Response Assays [mostly on tissues]• Graded responses to varying doses• Unknown dose response measured on same tissue

12

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.

13

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

14

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 estimateEg: histamine on guinea pig ileum, Posterior pituitary on rat uterus.

15

Interpolation assay.• Bioassays are conducted by determining the

amount of preparation of unknown potency required to produce a definite effect on suitable test animals/organs/Tissue under standard conditions. • This effect is compared with that of a

standard. Thus the amount of the test substance required to produce the same biological effect as a given quantity the unit of a standard preparation is compared and the potency of the unknown is expressed as a % of that of the standard by employing a simple formula.

16

Multi point Bioassay.• This method incorporates the

principle of interpolation and bracketing. • 2+1 indicates- Two response of

Standard and one response of Test respectively. • This procedure of 2+1 or 2+2 is

repeated 3 times or 4 times based on the method with crossing over of all the samples. • It can further divided as 3 point, 4

point and 6 point bioassay.

17

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)

18

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

19

Bioassays of some important drugs

Bioassay of insulin

• Standard preparation and unit: It is pure, dry and crystalline insulin. One unit contains 0.04082 mg. This unit is specified by Ministry of Health, Government of India and is equivalent to international unit. • Preparation of standard solution: Accurately weigh 20

units of insulin and dissolve it in normal saline. Acidify it with HCl to pH 2.5. Add 0.5% phenol as preservative. Add 1.4% to 1.8% glycerin. Final volume should contain 20 units/ml. Store the solution in a cool place and use it within six months.

20

Preparation of test sample solution: The solution of the test sample is prepared in the same way as the standard solution described above.

1. Rabbit Method: Selection of rabbits: They should be healthy, weighing about 1800-3000 gms. They should then be maintained on uniform diet but are fasted for 18 hrs. before assay. Water is withdrawn during the experiment. Standard and Sample Dilutions: These are freshly prepared by diluting with normal NaCl solution so as to contain 1 unit/ml. and 2 units/ml. Doses: The dose which can produce suitable fall in blood sugar level is calculated for the standard. Principle: The potency of a test sample is estimated by comparing the hypoglycemic effect of the sample with that of the std. preparation of insulin. Any other suitable method can also be used.

21

Experimental Procedure: Animals are divided into 4 groups of 3 rabbits each. The rabbits are then put into an animal holder. They should be handled with care to avoid excitement. First part of the Test: A sample of blood is taken from the marginal ear vein of each rabbit. Presence of reducing sugar is estimated per 100 ml. of blood by a suitable chemical method. This concentration is called ‘Initial Blood Sugar Level’. The four groups of rabbits are then given sc. injections of insulin as follows:

22

From each rabbit, a sample of blood is withdrawn up to 5 hrs. at the interval of 1 hr. each. Blood sugar is determined again. This is known as ‘Final Blood Sugar Level’.

Second part of the test (Cross over test) : The same animals are used for the second part. The experiment can be carried out after one week. Again they are fasted and initial blood sugar is determined. • The grouping is reversed, that is to say, those animals

which received the standard are given the test and those which received the test are now given the standard. Those animals which received the less dose of the standard are given the higher dose of the test sample and vice-versa. This test is known as ‘Twin Cross Over Test’.

• Mean percentage decrease in blood sugar of the first and second part is calculated.

23

Mouse Method: Mice show characteristic convulsions after s.c. inj. of insulin at elevated temperatures. The percentage convulsions produced by the test and standard preparations are compared.

Experimental procedure: Minimum 100 mice weighing between 18-22 gms. of the same strain are used. They should be maintained on constant diet. They should be fasted 18 hrs. prior to the experiment.

Standard and sample dilutions: Dilutions are prepared with sterile saline solution, so as to contain 0.064 units/ml. (std dilution I) and 0.096 untis/ml. (std. dilution II). Similarly, test sample solutions are also prepared. Mice are divided into 4 groups each containing 25 mice and insulin is injected s.c. as follows:

24

• The mice which convulse or die are taken out of the incubator and observed.

• Percentage convulsions produced by the test sample are compared with those of the standard sample.

25

Other bioassay’s.

• Immunological assay (ELISA).• Micro-bioassay.• Radioimmunoassay. • Biotechnology.

26

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.

27

Uses of Bioassay

• To measure the pharmacological activity of new/ chemically undefined substances

• To investigate the function of endogenous mediators

• To measure drug toxicity and unwanted effects

• To measure the conc of drugs and other active substances in the blood or other body fluids

•Determination of potency, ED50/LD50 of drugs

•New drug development

•Measure clinical effectiveness

28

29

DisadvantagesKey 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

30

Conclusion

• Successful tool in estimation & discovery of biologically active substances

• Sensitivity & Specificity – important tool in pharmacology

31