Burn and Dale enunciated certain principles for conducting bioassays. These include, o All bioassays must be comparative and compared against a standard drug or preparation. This is to overcome errors due to biological variation.
o The standard and the new drug should be identical to each other, so that their dose-response curves will have the same slope and would be parallel to each other, i.e., the potency ratio would be constant all along the response levels.
o The method for comparing the unknown and the standard should preferably test the therapeutic property of the drug. Ideally, an analgesic is tested for analgesic activity and an anti-convulsant for anti-convulsant activity. However, this is not always convenient or possible to do so. For e.g., For estimating digitaloid drugs, the cardiac arrest in pigeon or guinea pig is used as the end point; which is the toxic effect of the drug but not the therapeutic effect.
o The method should eliminate all possible errors and allow an estimation of the error due to biological variation in different animals/persons at any one time and in the same animal/person at different times. Precautions should be taken to minimize errors due to biological variations. These include the selection of suitable animals/preparations and also the selection of the experimental conditions.
o The results of the test should be subject to statistical analysis to minimize errors due to biological variation.
• Bioassays may be broadly classified into 2 types depending upon the type of
responses recorded, i.e.,
(A) Quantal Bioassays:
• The quantal bioassay is an all or none phenomenon. In this form of bioassay at least 2 groups of animals are employed. The response is either positive or negative, i.e., there is no intermediate response. For e.g.,
o Insulin induced hypoglycemic convulsive reaction, i.e., an animal receiving insulin either show convulsions or does not;
o The animal receiving a dose of a drug, as in toxicity studies, either dies or does
• This method of bioassay is not very accurate, but can be employed for the following cases:
o Comparison of threshold responses.
o Comparison of ED50 or LD50.
Concentration of test substance =
Threshold dose of Std.
Threshold dose of test × Concentration of Std.
Concentration of test substance =
ED50/LD50 of Std. ED50/LD50 of Test × Concentration of Std.
• Graded responses are those that are measured by continuous variables such as weight, body temperature, blood glucose level, blood pressure, the number and strength of contractions of heart, respiratory rate, the extent to which an isolated tissue contracts or relaxes etc., The graded responses may be assessed by using either the whole animal or a part of the animal.
• The graded bioassays are based on the observations that there is a proportionate increase in the observed response with a subsequent increase in the concentration or dose. Then the test responses are compared with that of the standard. The parameters employed in such bioassays are based on the nature of the effect of the substance that is expected to produce. For e.g., contraction of a smooth muscle preparation (guinea pig ileum) for assaying histamine; the study of blood pressure response in case of adrenaline.
• This type of assay gives almost identical results. The choice of the assay depends upon,
o Precision of assay demands.
o Quantity of the sample available.
o Availability of experimental animals.
• The various methods of graded bioassays are,
1. Matching Dose Bioassay:
• It is the simplest form of all graded bioassays and involves no calculations. In this type of bioassay, the response of the standard substance is recorded first and then the response of the test substance is tried to match with that of the standard by a trial and error process, until they produce equal effects. It is also called as the analytical dilution assay as the assay involves the determination of the factor by which the test substance is either diluted or concentrated in order to produce a response that is equal to that of a known amount of the standard drug. A corresponding concentration of the test substance is then calculated.
o This assay is generally employed when the ample amount of sample is available.
Since the assay does not involve the recording of CRC, the sensitivity of the
preparation is not taken into consideration.
o The assay does not depend on the assumption of a dose response relationship.
o Purely subjective method.
o Inefficient as preliminary effects are not utilized in final assessment.
o Lot of experimental errors, which cannot be determined.
o A crude method and not the exact method of determining the potency of a drug.
o Precision and reliability are poor.
Multiple Point Bioassays:
• The various types of multiple point bioassays, which are also based on dose-
response relationship, are:
o 2-point bioassay
o 3-point bioassay
o 4-point bioassay
o 6-point bioassay
o 8-point bioassay
• In these bioassays, the responses are repeated several times and the mean of each is taken. Thus, chances of error are minimized in these methods. The sequence of responses is recorded as per the Latin square method of randomization in order to avoid any bias.
(A) 2-Point Bioassay:
• Any one dose that produces from minimal to maximal response of the standard and the test substances are taken from the DRC and then their % of response is calculated.
Concentration of Test Substance =
% Response of Standard % Response of Test × Concentration of Standard