Biological assays/Bioassays

-A type of scientific experiment. Conducted to measure the effects of a substance on a living system and are essential in the development of new drugs and in monitoring environmental pollutants. or Determination of the chemical & biological agents such as the drugs, hormones, vitamins, toxins & anti-toxins by means of the biological indicators such as blood pressure, blood glucose, muscle contraction & the inhibition of the growth of the micro-organisms. -Animal, organ, tissue, cell lines/microbes or sub cellular parts are used for the purpose. -Can be divided into two categories: i)Qualitative (Measures the physical effects) e.g. abnormal growth or deformity etc. ii)Quantitative -Concentration or potency of a substance by measurement of the biological response that it produces. Conditions of usage: i)Determination of the drug potency. ii)Screening of the new pharmaceutical agents. iii)Determination of the therapeutic advantages of the one drug with another drug. iv)To establish the structural activity relationships. v)Determination of the pharmacological activities of the new drugs. iv)To determine the specificity of a compound to be used e.g. Penicillins are effective against Gram+ve but not on Gram-ve v)Chemical composition of samples are different but have same biological activity Or activity may vary even though the chemical is the same ( e.g. due to optical isomerism) No alternatives in the following conditions: i)For the drug whose active principles are unknown. (e.g. Extracts, crude drugs) ii)For the drugs chemical methods are not available. (e.g.Cyanocobalamin in some dosage forms)

iii)For the samples which are available in minute

quantities.

Principle:
It is to compare the sample substances with the international standard preparations of the same & to measure the potency of the sample that is required to produce the same level of biological effect as that of the standard drug.

Types of bioassays : i)Quantal assays(End point method) ii)Graded response assays

i)Quantal assays: -To draw or elicit an all or none response in the different animals such as the following: a)Digitalis induced cardiac arrest in the guinea pigs. b)Digitalis induced head drop in the rabbits. ii)Graded response assays: -Varying doses are given to one animal. -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.

Variation of bioassays based on biological systems:

i)Animal(mouse, rat, guinea pig, rabbits etc) ii)Micro-organisms or cell based assay (using microbes like bacteria, fungi or cultured cells for antibiotics & growth stimulants/suppressants. iii)Plant(using plant constituents to evaluate a sample)

i)Animal(mouse, rat, guinea pig, rabbits etc) Bioassays for agonists: a)End point method(based on quantal effect): -The threshold dose producing the therapeutic effect is measured on the each animal. -Then comparison is made between the average results of the two groups of animal. -Sp./Std. are carried out simultaneously. -Finally potency is calculated from the following formula: Conc. of the unknown= threshold dose of Std. X Conc. of Std. threshold dose of Sp. b)Matching method(based on the graded response): -The constant dose of the test is bracketed by varying doses of the standard until the exact match is observed between the sample & the standard. -Response of the standard is measured at 20% & 70% of the sample. -The test will be repeated by increasing or decreasing the standard doses until the three equal responses are obtained. The concentration of the sample is determined by the following formula: Concentration of the sample= dose of Std.xConc.of Std. dose of the Sp. c)Graphical method(based on the graded response): -5-6 responses of standard graded doses of the standard drug are taken & the two doses of the sample drug are taken. -The height of the peak is plotted against the log dose. -The dose of the sample is directly taken from the graph. Conc. of the Sp.= [n1/t] x antilog{[(T-S1 )/(S2 -S1)] x [log n2 / n1]} x Cs Where, n1 is the lower standard dose; n2 is the higher standard dose; t is the test dose, S1 is response of lower standard dose; S2 is the response of the higher standard dose, T is the response of test; Cs is the concentration of the standard.

ii)Micro-organisms or cell based assay: -It is used to evaluate the potency of the formulations used. (to follow purification process, its distrbution within the blood & tissues when given to animals & finally human. Monitor of the antibiotics in blood & urine in a trace amount) -Used in case of unavailability of physical or chemical method. -Is performed using a suitable test organism. -Most micro-biological assay procedures are highly sensitive(up to 0.1mcg) & selective. -Sensitivity & selectivity are to measure the characteristic activity of biological products. Assay techniques: i)Plate diffusion ii)Tube-dilution i)Plate diffusion: -The agar plates are inoculated with a test organism. -Measured amounts of test substance are introduced into agar plates. -It diffuses into the agar & produces zones of inhibition. -The diameter of the zones may be plotted against log potency or conc. for a standard. -By interpolation, potency of the test material can be determined. -Suitable for antibiotic potency test. ii)Tube dilution: -Suitable dilutions of sample are introduced into an appropriate nutrient medium. -Suppression or stimulation of growth takes place. -Suitable for the determination of vitamin B & amino acids. -Measurement is done by nephelometry, titration or gravimetry.

Applications: --------------------------------------------------------------------------------------------Drug name Activity assayed Experimental animal/microbes--------------------------------------------------------------------------------------------Digitalis Fall in BP Guinea pig blood pressure Adrenaline Rise in BP Blood pressure in the spinal cat Acetylcholine Contractile effect Isolated rectus of the frog Heparin Prolongation of the Blood clotting time Whole blood of OX Insulin Lower of the BL. Rabbits glucose level Oxytocin Ejection of milk Female rabbits Erythromycin Growth inhibition Bacillus pumilus/ Micrococcus luteus