What is Bioassay: Definition, Methods & advantages of Bio assays
What is Bioassay? Definition & principle of bioassay
Bioassay is an assay designed to analyse any compound by use of a suitable biological system like animals, tissues, microbes etc.
Bioassay definition: It is defined as 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.
In bionalysis the response produced by the test compound is compared with that of standard sample the way similar to other analytical methods but here the biological system is involved in the determination.
Principle of bioassay: The bioassay compares the test sample with a same Internationally applicable standard substance. It determines the quantity of test sample required to produce an equivalent biological response to that of standard substance.
Standard samples are accepted by expert committee at international level and they represent fixed units of activity.
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Why Bioassay? Advantages & Uses of bioassay
Bioassays have some different role and purpose over other assay techniques.
1. They not only help to determine the concentration but also the potency of the sample. (Potency is a term which denotes activity of the compound per molecule basis. i.e. if a compound shows better activity at minute concentration, greater is the potency, and if its activity is low at lower concentrations, lesser is the potency).
2.It is especially used to standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptics etc. as these are all used over biological system in some or other form.
3. These also help determine the specificity of a compound to be used ex: Penicillin's are effective against Gram+ve but not on Gram-ve. Testing of infected patients sputum helps determine which anti-biotic be given for quick recovery.
4. Certain complex compounds like Vitamin B-12 which can't be analysed by simple assay techniques can be effectively estimated by Bioassays.
5. Sometimes the chemical composition of samples are different but have same biological activity.
5. For samples where no other methods of assays are available.
Gives protocol, method and applications of Bio assay.
Bioassay Methods / types of bioassays
Basically there are two types of bioassays as per the technique used in determination of the Sample under test.
1. Graded Response Assay
2. End Point or Quantal Assay
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.
In the graded dose response relationship, relates the size of the response to the drug in a single biologic unit as the dose administered increased the pharmacological response also increases and eventually reaches a steady level called the ceiling effect there will be on further increase in response even with an increase in dose.
The graded dose response curve is obtained by plotting a graph with dose on the X-axis and response on the Y-axis. It is usually sigmoid in shape however the log dose response curve is almost a straight line and particularly useful in bio assay.
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.
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. Here the sample effect is identified by the response it produces on the biological system. Digitalis produces cardiac stimulation on further doses it produces cardiac arrest.
(+)d TC (Tubocurarine) producing neck relaxation in rabbit, Here as the sample is injected to the neck muscle of the Rabbit, the neck starts to droop. On further doses there is complete hanging of the neck and rabbit has no ability to lift the neck
Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is done by this method.
The direct bioassay videos on animals is unethical to be displayed. Hope you will understand.
Matching point 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
(The Rectus abdominus muscle is a skeletal muscle of frog commonly used to see the effect of cholinergic related drugs. The muscle is contractile and each contraction response is recorded as spike or peak as you can see in the videos. The videos are intended for your understanding of the concept of methods followed in bio-assay.)
a) Matching point or bracketing method
b) Interpolation assay
c) Three point (2+1) assay
d) Four- point (2+2) assay
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.
To determine potency of test a log dose response curve is plotted. Check the video for more.
Interpolation assay Here a log dose-response curve is plotted with the standard. The concentration of the test is then read from the graph.
Three point (2+1) & Four- point (2+2) assay: This method incorporates the principle of interpolation and bracketing. 2+1 indicates- Tow 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.
Here cross over method is used during the assay as.
S1 S2 T, T S1 S2, S2 T S1 doses.
Check the video on the left for more details.
Bioassay systems and techniques
The bioassay systems vary based on the biological system used like animals (mouse, rat, guinea pig, rabbits etc), plant bioassay (using plant constituents to evaluate a sample like(haemolytic activity) microbiological or cell based assay (using microbes like bacteria, fungi or cultured cells for anti biotic compound screening etc).
Based on techniques they can be differentiated into two broad types like
a) 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 rodents, rabbits etc.
b) 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
c) Ex vivo techniques: These techniques employ a tissue or cells of recommended living system to study the effect of compound under test in suitable conditions within the stipulated time of organ survival outside the body. The methods described in the videos employ a living tissue of an animal in an apparatus to study the contractile effect of drugs.
Ex: Use of any isolated organ from animals in a glass ware to study the effect of compound within the period of its survival outside the living body with provision of only oxygen, glucose and isotonic salts to maintain cell & cell organelles integrity.