BIOASSAY AND ITS TYPES
Introduction:- Bioassay is defined as estimation or determination of concentration or potency of physical, chemical or biological agents 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. An assay is a form of biological experiment; but the interest lies in comparing the potencies of treatments on an agreed scale, instead of in comparing the magnitude of effects of different treatments. Biological assays or biological standardizations or simply bioassays are methods used for estimation of the potency of substances by observing their pharmacological effects on living animals (in vivo) or isolated tissues (in vitro) and comparing the effect of these substances of unknown potency to the effect of a standard.
In this analysis 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. Bioassay is Assessment of a biological substance. Bioassay or biological standardization is a type of scientific experiment typically conducted to measure the effects of a substance on a living organism and is essential in the development of new drugs and in monitoring environmental pollutants.
Structure of Biological Assay:- The typical bioassay involves a stimulus applied to a subject. Application of stimulus is followed by a change in some measurable characteristic of the subject, the magnitude of the change being dependent upon the dose. The intensity of the stimulus is varied by using the various doses by the analyst.
Principle of Bioassay:- Active principle to be assayed should show the same measured response in all animal species. Bioassay involves the comparison of the main pharmacological response of the unknown preparation with that of the standard. The method selected should be reliable, sensitive, and reproducible and should minimize errors due to biological variation and methodology. The degree of pharmacological response produced should be reproducible under identical conditions. The reference standard and test sample should have same pharmacological effect and mode of action, so that their DRC curve run parallel and their potency ratio can be calculated. Activity assayed should be the activity of interest. Individual variations must be minimized/accounted for. Bioassay might measure a diff aspect of the same substance compared to chemical assay. The test solution and standard should be compared for their established pharmacological effect using a specified pharmacological technique. Prior to the development of sensitive, precise, accurate chemical and binding displacement assays for the presence or concentrations of drugs or autacoids we had to rely on bio-assays.
Types of Bioassays
There are three main types of bioassays (other than qualitative assays):-
- Direct Assays
- Indirect Assays based upon quantitative responses
- Indirect Assays based upon Quantal responses (“all or none”)
- Direct Assay:- Doses of the standard and test preparations are sufficient to produce a specified response, and can be directly measured.
- Indirect Assay:- In indirect bio-assays the relationship between the dose and response of each preparation is first ascertained. Then the dose corresponding to a given response is obtained from the relation for each preparation separately.
- Quantal Assay:- This response is in the form of “all or none” means no response or maximum response. These can be biossayed by end point method. Predetermined response is measured which is produced by threshold effect. Quantal Responses are population response based on an all-or-nothing (0 or 1 – presence or absence) response such as death.
Concentration of unknown = Dose of the standard × Concentration of standard/ Dose of test
- Graded Assay:- It is proportional to the dose and response may lie between no response and maximum response. Graded Responses can be any type of measured responses in isolated tissues in particular, but also in whole animals. Such responses are infinitely graded and there are a large number of them.
Examples – contractions of muscle, blood pressure, blood sugar concentrations, etc.
- Matching Method:- In this type of assay the test substance and the standard are applied and the responses obtained are matched by a trial and error process until they produce equal effects. This may also limit to analytical dilution assay, as the assay involves the determination of the factor by which the test substance is diluted or concentrated in order to produce response that is equal to that of known amount of the standard drug. Its advantage is that it does not depend on the assumption of a dose-response relationship.
- Bracketing Method:- Bracketing bioassay is performed by selecting two standard doses, which will give a close bracket on either side of the response produced by the unknown. The working dose of standard is first determined in the sensitive part of dose-response curve, that is, a dose that will approximately produce 50% of the maximal concentration. The dose of the standard drug is kept constant throughout the experiment, in order to have some idea about the change in the sensitivity of tissue with time. The standard drug is added at fixed intervals but alternating with the test so that each response produced by a dose of test substance is bracketed by responses produced by the dose of standard. The response of test substance is bracketed between two responses of the standard. Close bracketing gives more accurate results.
- Interpolation Method:- This is a simplest form of graded response assay and involves no statistical data and many calculations. In this assay the dose response curve is fist obtained from different doses of standard ach solution. The concentration of unknown is then read from the standard graph. Interpolation method of bioassay is less time consuming and yet reliable compare to matching type of bioassay. One of the main advantages of this essay is that the sensitivity of the tissue is first determined by prior plotting of a dose response curve with a known agonist as in the case with acetylcholine. If the linearity of curve is good, one can do very accurate estimate of the test substance unknown sample.
- Three Point Bioassay:- In three point bioassay, the DRC of standard & test samples is first obtained from the responses due to graded doses. From the DRC of standard, two standard doses are selected in such a way that they have produced 25% & 50% of the maximal response respectively & are designated as S1 & S2. The responses of these doses lie on the steepest & straightest part (linear) of the curve. From the DRC of test sample one test is selected such that it gives a response which lies in between the two standard responses that is it gives a greater response than S1 & a smaller response than S2 & is designated as T. After selecting the standard & test doses, the bioassay is performed by recording the standard & test responses in randomized fashion as per Latin square design. The pattern of addition of doses is S1, S2, T; S2, T, S1 & T, S1, S2 in 3 successive cycles. The mean values of height of contraction for all the 3 doses are calculated and are used in plotting the graph so as to estimate the potency of the test sample. The precision and reliability of this method is much better than matching and bracketing methods of bioassay & the sensitivity of the isolated tissue preparation is assessed prior to testing the unknown sample.
- Four Point Bioassay:- The classic 2X2 parallel assay involves being able to measure parallelism where drugs acting through the same mechanism are expected to produce parallel dose-response curves.
- Seed Bioassay:- Seeds are living organisms that may be harmed by chemicals. The seed bioassay technology has been implied as decisive and lab scale and method to assess the toxicity of any substance on profitable crops. The seedling germination and seedling growth impressions under contrasting concentrations of industrial derivation can give some perception about the abolishing or toxicological impact of industrial effluents on plants. A lab-scale bioassay of distillery effluent was conducted using few profitable cereal crops in order to seed the feasibility of utilizing distillery effluent for crop irrigation purposes.
- Antimicrobial Assay:- Standard and clinically isolated microorganism strains were used for antimicrobial assays. A large number of human, animal and plant disease are caused by pathogenic microbes. Infection due to fungi and bacteria has been a major cause of death in higher organisms. The microbial assay for antibiotics is a method that uses microorganisms to determine the antimicrobial potency of the antibiotics contained in medicine.
- Antifungal Assay:- Fungal infections have been reported to have dramatically increased in the past decade, and these often occur as systemic infections or as co-infections with other diseases, such as AIDS or cancer, or in patients who are immuno-compromised. There is considerable need to discover new fungi-toxic compounds in view of the many plant and human fungal diseases. Some of the common plant fungal diseases are potato late blight, tobacco blue mould; hop downy mildew, Dutch elm disease, ergot of rye, cereal rusts, corn blight and grape downy mildew. The human fungal diseases include athlete‟s foot, Aspergillosis, Actinomycosis, Histoplasmosis and Corcidiomycosis. The rapid increase in fungal infections and the growing number of new antifungal agents indicate an increasing need for rapid and accurate methods for antifungal screening and susceptibility testing. Some fungi can be beneficial to man since they attack harmful insects. The two main methods includes:-
- Cylinder Plate Method
- Paper-disc Method
- Antimitotic Assay:- Antimitotic agents are defined as any applied stimulus which produces a consistent change/deviation in the mitotic cycle. Inhibition of cell division is a measure of the antimitotic activity of chemical compounds. Antimitotic chemical compounds such as vinblastine and podophyllotoxin have been shown to inhibit cell division of fertilized sea urchin eggs and starfish oocytes. Normally the reaction should be reversible with time, removal of stimulus or on the addition of an antagonist.