BIOASSAY ANSWERS (SET 1

)
Bioassay Definition
Estimation of the concentration /or potency of a substance by measurement of
the biological response that it produces.
Uses:
1. To measure the pharmacological activity of new or chemically undefined
substances.
2. To investigate the functions of endogenous mediators.
3. To measure drug toxicity and unwanted effects ED50 and LD50.
4. Drug discovery: the ability of extracts of posterior lobe of pituitary to
produce rise in BP and contractions of uterus was observed in 1930s and
were found to be ADH and Oxytocin.
AIM: Comparing the activity of 2 preparations, a standard(s) and an unknown(u)
on a particular preparation.
Bioassay must provide an estimate of dose/concentration of unknown that will
produce the same biological effect as that of known dose/concentration of
standard.
Main problem with bioassay: biological variation.
M= potency ratio between S/U

M
Response
U

S

Log vol

In practice, most bioassays will give
results with 5% confidence limits lying within ±20%.
Graded response / Assay
E.g.: change in BP, glucose concentration, concentration of strip of smooth
muscle.
The steepness of the DRC is a property of the test system and has nothing to do
with biological variation
Relates to size of responses in single biological unit.
All or none response / Discontinuous / Quantal
E.g.: death, loss of righting reflex
LD50, ED50, Polygenic Random variation
As the dose increases the response increases reflecting the extent of receptor
occupancy.

Error is distributed all through the graph independent of dose. ii. but both have similar activity. Eg : [5] What is comparative assay?  Here the test compound is chemically different from the standard. Eg: If we are preparing tetanus antitoxin then the activity of 1TU of our antitoxin should be standardised with that of standard preparation in National Serum Institute. Several preparations can be tested from a single animal. Relatively small amount of test material required. What is bio standardisation? Comparison and adjustment of the strength of the sample with that of the standard under controlled conditions. M . Biostandardisation = Alteration) Why Log Phase Response? Results can be plotted (obtained) even with wide variation of doses over a 1000 fold range. Drug effect is tested directly without factors like A. iii. E [2]      Why ileum? Relatively more resistant to trauma/handling Easy to set up Produce larger contractions than in duodenum and jejunum Less mesentery More receptors. D. [1]    Advantages of isolated tissue. Eg: Histamine and Acetylcholine on G. Here it is the environment within the inner organ bath with tissue. The middle portion of sigmoid is almost linear. [3] What is Bio phase?  Environment in which drug interacts with receptor without intervening diffusion barriers. [4] What do you mean by analytical dilution assay?  Here the nature of test compound and the standard are same and the concentration / potency of test is determined by dilution or concentration with standard. physiological salt solution and drug. Necessary to regulate doses of crude extract.Threshold dose and ceiling dose This allows us to compare therapeutic efficacy of various compounds.pig tracheal chain [6]     [7] i.1547 mg) (Bioassay = Measurement.Copenhagen (0. .

Higher the pD2 value. [12] Uses of salts in physiological salt solutions: Na+ -carry the depolarising current essential for excitation.iv.  Timing of doses not taken into account.  Purely subjective. It is the mean negative logarithm of molar concentration producing 50%of maximal response. [10] Drugs that contract and relax guinea pig ileum? Contraction Relaxation/No effect *5 HT *Noradrenaline *Histamine *Adrenaline *Acetylcholine *Angiotensin *Bradykinin [11] Disadvantages of Matching Assay:  It does not consider changes in tissue sensitivity with respect to time. .It was first designed by Rudolph magnus in 1904  The present day organ bath was designed by Sir Henry Dale.  It is based on application of few doses hence cannot be subjected to statistical evaluation (error cannot be calculated). Higher is potency. What is pAx value? Schild proposed it to express drug antagonism.  Student organ bath having two units of inner tissue bath (double unit organ bath) was designed by Gaddum. Negative log of molar concentration of an antagonist. When two are more graphs are plotted middle portions will be almost parallel hence can be compared easily. [8]    [9]   What is pD2 value? Another way of expressing potency. ions -provide adequate osmotic pressure K+ ions -essential for bringing the process of relaxation to completion Ca+ ions -for coupling the excitation with contraction -stabilises membrane potential Mg+ ion -reduce spontaneous contractility in mammalian tissue NaH2PO4 -acts as buffer [13] Who discovered organ bath?  Organ bath is an assembly for recording contractions of isolated tissue. which will reduce the effect of a multiple dose X of an active drug to that of single dose.

due to vasoconstriction. [22] Give an example of indirect bioassay?  If the potency of sample is estimated by comparing its log dose response with that of standard then it is called indirect assay. [16] What are the uses of Aeration?  Provides oxygen to the tissue.4. The colour .  Prolonged aeration tends to alter pH  At lower pH the tonus of the tissue decreases. when injected in the white leghorn cock.3-7. [18] How will you manage Spontaneous Activity of tissue?  For frog rectus: 5-10 µg/L of neostigmine can be added to the reservoir (salt solution)  For guinea pig ileum: o Atropine 1mg/L or o Adrenaline (But there will be baseline shift) or o Lowers the bath temperature [19] How will you prevent the friction between lever and drum ?  The writing point should be sharp  The paper should be smooth  Smoking should be thin and uniform  Vibrator can be fixed [20] Give example of microorganism used for bioassay.  Facilitates diffusion of drug  Stirring. hence all records to be read from left to right.  Euglena gracilis is used for vit B12 bioassay. there by altering the effect of drugs. there is bluish discolouration of the comb. [21] Examples of isolated cells in bioassay  Isolated Leydig cells of testes are used in-vitro for bioassay of LH hormone.  Speed of drum o *Guinea pig ileum-32 minutes per revolution o *Frog rectus-128 minutes per revolution [15] How will you check pH? Why is it important?  The pH of salt solutions should be maintained between 7. Plasma LH has ability to stimulate testosterone synthesis. [17] What is Differential bioassay?  If more than one active substance is present then it is called Differential Bioassay.  Ph is checked with Litmus paper or commercially available pH strips.[14] Information about drum  It is called Sherrington Drum  By convention.  Maintains vitality of tissue and helps to overcome inertia. it rotates in a clockwise direction.  Old ex: ergot preparation.

Substance 1. Chemical assay is too complex or insensitive c.d-TC 4.intensity varies with the dose and this effect may be employed for bioassay of crude ergot preparation. Assay Assay: Estimation of amount or activity of an active principle in unit quantity of preparation Eg: Chemical assay.Vasopres sin 2. e) Problems of individual variation must be minimised or taken into account. d. Requirements:  High sensitivity  Specificity Eg: G.D Preparation Increases BP in anaesthetised rat Decreases urine output in hydrated rat. Active principle is unknown /cannot be isolated easily.Vit. Principles: a) Effect produced should be same in all animal species. Q.Insulin 3. immunological assay Indications: a. Whole Animal Bioassays.Estrogen 5. b) Certain quantity of drug produces same degree of pharmacological responses in the same animal provided other conditions remain constant c) Reference standard must owe its activity to the principle for which the sample is being bioassayed. Chemical composition not known but substance has special biological action.pig contracts with Acetylcholine and histamine. d) The activity assayed should be the activity of interest. b. therefore the tissue has be atropinised  Reproducibility  Accuracy  Stability: Tissue /animal should be bioassay fit for long time. Hypoglycemic convulsion or death in mice Head drop in rabbit due to paralytic relaxation of skin of the neck Vaginal cornification in ovariectomised female rat Alleviation of rachitic state in rat Q. biological assay. Indirect assay: potency of the sample is estimated by comparing DRC of Standard Direct assay: ED 50 and LD 50 calculated Eg: digitalis bioassay in guinea pig . Eg: long acting thyroid stimulator. Q. Drugs differ in composition but have same pharmacological action eg: digitalis glycosides from various sources. Sat.