BIOLOGICAL ASSAY OF ANTIBIOTICS , VITAMIN D , DIGOXIN & INSULIN

BIOLOGICAL ASSAYS CHAPTER NO 6
PREPARED BY :HASNAT TARIQ (GDIP) 1
6. BIOLOGICAL ASSAYS
ASSAY
Assay refers to the quantitative or qualitative analysis of a compound. Amount or activity of an active principle in
unit quantity of preparation.
An assay is an investigative (analytic) procedure in laboratory medicine, pharmacology, environmental biology,
and molecular biology for qualitatively assessing or quantitatively measuring the presence or amount or the
functional activity of a target entity (the analyte) which can be a drug or biochemical substance or organic sample.
Types of Assays:
1.Chemical Assay:
It is the study of the separation, identification, and quantification of the chemical components of natural
and artificial materials.
2.Immunoassay:
A technique that makes use of the binding between an antigen and its homologous antibody in order to
identify and quantify the specific antigen or antibody in a sample.
3.Biological Assay
Potency or concentration of an active principle in unit quantity of preparation by measuring its biological
response on living tissues
Comparison Of Chemical & Bioassay
Bioassay Chemical Assay
1. Difficult to handle 1. Easy to handle
2. More men power Required 2. Less men power required
3. Active constituent &
structure not known.
3. Active constituent & structure
fully established.
4. More expensive 4. Less expensive
5. More time consuming 5. Less time consuming
6. More sensitive 6. Less sensitive
7. Less Precise 7. More Precise
BIOLOGICAL ASSAY
A bioassay is a procedure for determining the concentration, purity, and/or biological activity of a substance (e.g.,
vitamin, hormone, plant growth factor, antibiotic, or enzyme) by measuring its effect on an organism, tissue, cell,
enzyme, microorganisms or receptor preparation compared to a standard preparation.
 Drugs which are primarily of natural origin are assayed by biological methods.
 The accuracy of biological assays is considered excellent if in the range of ± 10%. However, chemical
assays can be expected to give a 100% accurate value.
Why we use bioassay?
It is usually advisable to use chemical assay alternatives where possible, yet, biological methods of assaying are
employed in the following circumstances:
1. If the chemical structure and identity and structure of the active principle in the drug has not yet been
fully comprehended.

2. If no adequate chemical assay has been devised for the active principle, although its chemical structure
has been established.
3. If the drug is composed of a complex mixture of substances of varying structure and activity.
4. If purification of crude drug, sufficient for performance of a chemical assay, is impractical or impossible.
5. If the chemical assay is not a valid indication of biological activity (e.g., due to lack of differentiation
between active and inactive isomers)
Standard preparations and units of activity
Bioassays are conducted by determining the amount of a preparation of unknown potency required to produce a
definite effect on suitable test animals or organs under standard conditions as compared to that of the activity of a
standard preparation. Standard preparations are used to minimize the source of error resulting from animal
variations.
 A standard preparation of the drug in question is used along with the drug on a similar set of test
animals under similar conditions, thus, eliminating, or at best minimizing the errors presented due to
animal variability. The standard preparations are a selected representative of the sample is question based
on national or international rules of standardization. Local laboratories make their own standards, to be
prepared and standardized against the national or international standard preparations.
 When units are referred to in the official biological assays and tests, the Unit for a particular substance is,
the specific biological activity contained in such an amount of the respective Standard Preparation. The
„Standard Preparation‟ refers to the standards used in that particular country with respect to its national
law or international following of standards.
Types Of Standard Preparation
Two types of standard preparation:
1) International standard and reference standard
USP units (highly recognized-able and authorized standard)
2) British standard and reference standard
Country wants to have its own standard preparation, then used according to its own law. For specific biological
activity small quantity of standard preparation are used
Potency
Measurement of drug activity expressed in terms of amount required to
produce an effect of given intensity.
Examples:
Highly potent drugs like morphine, alprazolam, chlorpromazine etc.
produced high response at low conc. And low potent drugs like ibuprofen and
acetylsalicylic acid produced low response at low conc.
Classification of bioassays
Bioassays are classified into three types based on the type of the effect produced. The result of the biological
assays can be:
1) QUANTAL
All or none response in all individuals.
E.g., Digitalis induced cardiac arrest in guinea pigs, hypoglycemic convulsions in mice by insulin.
Calculation of LD50 in mice or rats.
Employed for:
• Comparison of LD50 and ED50
• Comparison of Threshold response

2) GRADED BIOASSAY
Graded response - response is proportional to the dose and response may lie between no response and
the maximum response.
 Types:
• Bracketing /direct matching
• Interpolation
• Multiple point assays
Three-point assay
Four-point assay
Six-point assay
• Cumulative dose response
E.g., Contraction of smooth muscle preparation
3) Characterized by developing in a measured period of time (e.g., the curative response of thiamine)
Advantages of bioassays
Following are the advantages of biological assays over chemical assays:
1. A biological assay determines the actual biological activity of a substance and not just its concentration
in a given sample.
2. The inhibitory influences of variations of the active principle in the dosage from or the physical state are
not reflected in the chemical assays, whereas, the biological assays account for it.
3. They not only help to determine the concentration but also the potency of the sample.
4. It is especially used to standardize drugs, vaccine, toxins or poisons, disinfectants, antiseptics etc. as
these are all used over biological system in some or other form.
5. 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 is
given for quick recovery.
6. Certain complex compounds like Vitamin B-12 which can't be analyzed by simple assay techniques can
be effectively estimated by Bioassays.
7. Sometimes the chemical composition of samples is different but have same biological activity.
8. For samples where no other methods of assays are available.
9. Biological products like toxin, anti-toxin, sera can be conveniently assayed. Measure minute (Nano mole
& Pico mole) quantities of active substances can detect active substance without prior extraction or other
treatment.
Disadvantages of bioassays (Limitations of bioassays)
Following are the disadvantages of biological assays over chemical assays:
1. Biological assays are less accurate
2. Biological assays are more time consuming
3. Biological assays can be more expensive
4. Biological assays are inconvenient and difficult to perform
5. Ethical obligations
6. Key problem is variability in response
7. Large number of animals to be used
8. Expertise in experimental design, execution of assay & analysis of data required
9. Time related changes in sensitivity of test organ.

10. Tachyphylactic responses of substance being assayed.
Indications Of Bioassay (Purpose of bioassay)
1. Chemical method is either;
 Not available,
 If available, too complex,
 Insensitive to low doses e.g., Histamine.
2. If active principle of drug is not known e.g., insulin.
3. Unknown Chemical composition, e.g., long-acting thyroid stimulator.
4. Chemical composition of drug differs but has same pharmacological action e.g., cardiac glycosides.
5. Active principle cannot be isolated e.g., posterior pituitary extract, insulin etc.
6. Biological activity of drug cannot be defined by a chemical assay e.g., Cis and Trans form of methyl
phenidate.
7. Not possible to separate interfering substance e.g., Vitamin D.
Principles Of Bioassay
1. To compare the test substance with the International Standard preparation of the same
2. To find out how much test substance is required to produce the same biological effect, as
produced by the standard
3. Activity assayed should be the activity of interest
4. Standard & test sample - similar pharmacological effects & mode of action
5. Both should be compared for their established pharmacological effect using specified technique
a. Ex: *Ach – contractile response on frog rectus *Histamine – contractile response on
guinea pig ileum
Problem of biological variation must be minimized
1. Experimental conditions - kept constant
2. Animals - same species, sex and weight
3. Number of animals - large enough to minimize error (individual variation)
4. Isolated preparations – sensitive
Characteristics of a good assay method
1. Sensitivity
2. Specificity
3. Repeatability
4. Reproducibility
5. Precision
6. Accuracy
7. Stability – tissue has to stay “bioassay-fit
Bioassay can be performed
In Vivo : Intact Animals
In Vitro : isolated tissues, organisms and specified cells
WHOLE ANIMALS
• Nor Adrenaline – Spinal Cat
• Cardiac Glycosides – Guinea Pig
• Insulin – Mice
• Estrogens – Ovariectamised Female Rat

MICRO ORGANISMS
• Vit.B12 – Euglena gracilis
• Tetracycline - Bacillus pumilus
ISOLATED TISSUE
• Acetyl Choline – Frog Rectus Abdominus muscle
• Histamine – Guinea Pig ileum
• Adrenaline – Rat uterus
• Oxytocin – Rat uterus oestrogen primed
DISPERSED CELLS
• Plasma LH (Luteinizing hormone) estimation by stimulation of testosterone synthesis - on isolated Leydig
cells
Bioassay of Antibiotics
Antibiotics
Substance (such as penicillin) that destroys or inhibits the growth of other pathogenic microorganisms and is
used in the treatment of external or internal infections.
While some antibiotics are produced by microorganisms, most are now manufactured synthetically.
Classification of antibiotics:
Antibiotics are classified in several ways.
1. On the basis of mechanism of action
2. On the basis of spectrum of activity
3. On the basis of mode of action

On the basis of mechanism of action:
On the basis of spectrum activity:
• Broad spectrum antibiotics:
– Amoxicillin
– Tetracycline
– Cephalosporin
– Chlorampenicol
– Erythromycin
• Short spectrum antibiotics:
– Penicillin –G
– Cloxacillin
– Vancomycin
– Bacitracin
– Fluxacillin
On the basis of mode of action:
• Bacteriostatic antibiotics
– Tetracycline
– Chlorampenicol
– Erythromycin
– Lincomycin
• Bacteriocidal antibiotics
– Cephalosporin
– Penicillin
– Erythromycin
– Aminoglycosides
– Cotrimoxazole

Antibiotics indications:
• Pneumonia
• Sepsis
• Meningitis
• Osteomyelitis
• Urinogenital Infections
• Gall Infections
• Scarlet Fever
• Mucous Membrane Infections
• Diphtheria
• Siberian Ulcer
• Gynecologic Infections
• Syphilis
• Respiratory Infections
• ENT Infections
• Fungous Infections
• Skin Infections
MICROBIOLOGICAL ASSAY OF ANTIBIOTICS
• The inhibition of growth under standardized conditions may be utilized for demonstrating the therapeutic
efficacy of antibiotics.
• Any subtle change in the antibiotic molecule which may not be detected by chemical methods will be
revealed by a change in the antimicrobial activity and hence microbiological assays are very useful for
resolving doubts regarding possible change in potency of antibiotics and their preparations.
PRINCIPLE
• The microbiological assay is based upon a comparison of the inhibition of growth of micro-organisms by
measured concentration of the antibiotics to be examined with that produced by known concentrations of
a standard preparation of the antibiotic having a known activity.
Two general methods are usually employed: -
A. The cylinder-plate (or cup-plate) method.
B. The turbidimetric (or tube assay) method.
PREPARATION OF MEDIA:
The Media required for the preparation of test organism are made from the ingredients.
• Minor modifications of the individual ingredients may be made, or reconstituted dehydrated media may
be used provided the resulting media have equal or better growth-promoting properties and give a similar
standard curve response.
• Dissolve the ingredients in sufficient water to produce 1000 ml and add sufficient 1M Sodium hydroxide
or 1M Hydrochloride acid, as required so that after sterilization the pH is bw 6.5 to 7.5.
PREPARATION BUFFER SOLUTIONS
• Buffer solutions are prepared by dissolving the following quantities of dipotassium hydrogen phosphate
and potassium dihydrogen phosphate in sufficient water to produce 1000 ml after adjusting the pH with 8
M phosphoric acid or 10M potassium hydroxide.
Buffer
No.
Dipotassium
Hydrogen
Phospahate,
K
2
HPO
4
(g)
Potassium
Dihydrogen
Phosphate,
KH
2
PO
4
(g)
pH adjusted after
sterilisation to:
1 2.0 8.0 6.0 + 0.1
2 16.73 0.532 8.0 + 0.1
3 - 13.61 4.5 + 0.1
4 20.0 80.00 6.0 + 0.1
5 35.0 - 10.5 + 0.1*
6 13.6 4.0 7.0 + 0.2

PREPARATION OF THE STANDARD SOLUTION
• To prepare a stock solution, dissolve a quantity of the Standard Preparation of a given antibiotic,
accurately weighed and previously dried where so indicated in Table, in the solvent specified, and then
dilute to the required concentration as indicated. Store in a refrigerator and use within the period
indicated.
PREPARATION TEST ORGANISMS
• The test organism for each antibiotic is listed in Table, together with its identification number in the
American Type Culture Collection (ATCC) and the National Collection of Type Cultures (NCTC) or the
National Collection of Industrial Bacteria (VCIB).
A. CYLINDER-PLATE OR CUP-PLATE METHOD: (Petri-Dish method)
• Inoculate a previously liquefied medium appropriate to the assay, with the requisite quantity of
suspension of the microorganism, add the suspension to the medium at a temperature between 40° and
50° and immediately pour the inoculated medium into the petri dishes or large rectangular plates to give a
depth of 3 to 4 mm.

• 4-8 sterile cylinders of 10mm height and 6mm inside diameter approx., made up of glass, porcelain,
aluminium are placed on the surface of inoculated medium. Instead of cylinders, 5-8mm holes can be
bored in the medium using sterile borer or disc can be used.
• Ensure that the layers of medium are uniform in thickness, by placing the dishes or plates on a level
surface. The inoculated plates are allowed to dry for 30min at room temperature or solidified in
refrigerator.
• Using the appropriate buffer solutions, prepare solutions of known concentrations of the antibiotic to be
examined
• Apply the solutions to the surface of the solid medium in sterile cylinders or in cavities prepared in the
agar. The volume of solution added to each cylinder or cavity must be uniform and sufficient almost to
fill the holes when these are used.
• Leave the dishes or plates standing for 1 to 4 hours at room temperature or at 4°, as appropriate, as a
period of pre-incubation diffusion to minimise the effects of variation in time between the applications of
the different solutions. Incubate them for about 18 hours at the particular temperature.
• Accurately measure the diameters or areas of the circular inhibition
zones and calculate the results.
L = 3a + 2b + c – e: H = 3e + 2d + c- a
5 5
Where
L = the calculated zone diameter for the lowest concentration of the
standard curve response line.
H = the calculated zone diameter for the highest concentration of the
standard curve response line.
c = average zone diameter of 36 readings of the reference point standard
solution.
a, b, d, e = corrected average values for the other standard solutions, lowest to highest concentrations,
respectively.

B. TURBIDIMETRIC OR TUBE ASSAY METHOD
• The method has the advantage of a shorter incubation period for the growth of the test organism (usually
3 to 4 hours) but the presence of solvent residues or other inhibitory substances affects this assay more
than the cylinder plates assay.
• Procedure
1. Prepare five different concentrations of the standard solution for preparing the standard curve by diluting
the stock solution of the Standard Preparation of the antibiotic & increasing stepwise in the ration 4:5.
2. Select the median concentration & dilute the solution of the substance being examined (unknown) to
obtain approximately this concentration.
3. Place 1 ml of each concentration of the standard solution and of the sample solution in each of the tubes
in duplicate.
4. To each tube add 9 ml. of nutrient medium, previously seeded with the appropriate test organism.
5. At the same time prepare three control tubes, one containing the inoculated culture medium (culture
control), and another identical with it but treated immediately with 0.5 ml of dilute formaldehyde
solution (blank) and a third containing uninoculated culture medium.
6. Place all the tubes, randomly distributed or in a randomized block arrangement, in an incubator or water-
bath and maintain them at the specified temperature, for 3 to 4 hours. After incubation add 0.5 ml
of dilute formaldehyde solution to each tube. Measure the growth of the test organism by determining
the absorbance at about 530nm of each of the solutions in the tubes against the blank.
L = 3a + 2b + c – e : H = 3e + 2d + c- a
5 5
Where
L = the calculated absorbance for the lowest concentration of the standard response line.
H = the calculated absorbance for the highest concentration of the standard response line.
a, c, b, d, e = average absorbance values for each concentration of the standard response line lowest to highest
respectively.

Standard preparations and units
The standard preparations are supplied as dry powders in sealed ampoules. The potency of an antibiotic is usually
describes as the units contained in 1mg of the powder of the standard preparation or as the milligrams of the
standard antibiotic powder that contains 1 unit.
The standard preparations and units for some antibiotics are given in the following table;
Bioassay of VITAMIN D
VITAMIN D (Anti-rachitic Vitamin)
1. Vitamin D is fat soluble vitamin.
2. It maintain the adequate plasma level of calcium in the body.
3. Deficiency results in rickets in children and osteomalacia in adults
4. Absorb calcium and phosphate in body and maintain mineralization of bone.
5. 7-dehydrocholestrol is a precursor and is converted into vitamin D3 in the skin in the presence of sunlight.
DEFINITION:
• Biological assay of vitamins is a type of biological assay performed with the aid of microorganisms.
• Many therapeutic agents, which either inhibit the growth of microorganisms or are essential for the
growth of them are standardized by biological assay.
PRINCIPLE:
• The activity of a preparation of antirachitic vitamin (vitamin d) is determined by comparing its
antirachitic activity with that of standard preparation of antirachitic( vitamin D) by a suitable method.
MATERIAL REQUIRED:
Standard Preparation and Unit:
• The standard preparation is the second international standard for Vitamin D3, established in 1949and
consisting of activated crystalline 7-Dehydrocholestrol. It is supplied in bottles as a solution in
vegetable oil (Cottonseed oil) containing 1000 units per gram (containing 0.025 mg of 7-
dehydrocholesterol); fresh solutions are dispensed every six months.
• The unit is contained in 0.000025mg of pure substance.
Subject: Very young rats (litters)

Procedure
1. Preliminary Period
2. Depletion Period
3. Rachitogenic Diet
4. Assigning Rats to Groups for Assay Period
5. Administration of Standard and Sample Doses
6. Assay Period
7. Line Test
8. Observations and Acceptability
1. Preliminary Period
30 days period and extends from birth to the first day of the depletion period
 During the preliminary period, use a dietary regimen that provides for normal development but is
limited in its content of vitamin D.
 At the end of the preliminary period, reject any rat that weighs less than 44 g or more than 60 g, or that
shows evidence of injury, disease, or anatomical abnormality
 Not fewer than forty young rats of either sex, shortly after being weaned, are used for the test. The
weight of the heaviest rat in a litter not exceeding that of the lightest by more than 10g
2. Depletion Period
 Depletion period, which extends from the end of the preliminary period to the first day of the assay
period.
 Provide each rat with the Rachitogenic Diet and water
3. Rachitogenic Diet
 They are fed for about 3 weeks on a rachitogenic diet, which
consists mostly of carbohydrates, proteins and electrolytes
and no fats. The Rachitogenic Diet consists of a uniform
mixture of the following ingredients in the proportions shown
in the accompanying table.
4. Assigning Rats to Groups for Assay Period
 Consider a litter suitable for the assay period when individual
rats in the litter show evidence of rickets such as enlarged joints and a distinctive wobbly, rachitic gait,
provided that the depletion period is not less than 19 or more than 25 days.
 The presence of rickets may be established also from the width of the rachitic metaphysis upon X-ray
examination of the proximal ends of the tibia or the distal ends of the ulna or by applying the Line Test
(described below) to a leg bone of one member of each litter.
 Record the weight of each rat, and assign it to a group
 Litters are divided into 4 groups each containing 7 or more litters. (One rat from each litter set of four or
two from each litter set of eight, being assigned to each group)
5. Administration of Standard and Sample Doses
 The litters in two reference groups receive x and nx doses respectively. (Where n is suitable value such as
2 or 3), respectively, of the standard preparation, while the rats of the other two groups receive,
respectively, doses of preparation being examined in the same ratio as the doses of the standard
preparation.
 Suitable doses of standard preparation may vary from 2 to 8 units. (x=2 units; n=1, 2, 3,4)

 Each rat may receive the whole of its dose at once or the dose may be divided into 8 daily doses. 10-14
days after receiving the doses of standard preparation or of the preparation being examined if given in
one amount, or ten to fourteen days after receiving the first fraction of the dose.
 Ratio of larger to smaller dose not less than 1.5 or more than 2.5.
 Before feeding, the Reference Standard and/or sample may be diluted with cottonseed oil, provided that
not more than 0.2 mL is fed on any one day.
 Store the oil solutions in well-closed bottles, protected from light, at a temperature not exceeding 10°,
and use within 5 weeks.
6. Assay Period
 Assay period last for 7-10 days.
 Throughout the assay period, maintain as uniform environmental conditions as possible for all rats, and
exclude exposure to antirachitic radiations.
 At the end of a fixed period of 7 to 10 days, weigh and kill each rat.
 Dissect out one or more leg bones for examination by the Line Test.
7. Line Test
 To stain the bones, the distal ends of the ulnae and radii may be removed, immersed for twenty-four
hours in a 4% w/v solution of formaldehyde in water
 Remove the proximal end of a tibia or the distal end of a radius, and clean adhering tissue from it, in any
one assay using the same bone from all animals.
 Rinse in purified water, immerse immediately in 1.5%w/v solution of silver nitrate for a few minutes and
rinse again in purified water.
 Expose the cut surface of bone, in water, to daylight or another source of actinic light until the calcified
areas develop a clearly defined stain without marked discoloration of the uncalcified areas.
 The staining procedure may be modified to differentiate more clearly between calcified and uncalcified
areas.
8. Observations and Acceptability
 Calcified and uncalcified areas are observed and relative potency of standard and sample is calculated.

 Score the degree of calcification of the rachitic metaphysis in each rat, according to a scale that allows the
average response to be plotted as a straight line against the logarithm of the dose. The result is calculated
by standard statistical method
Bioassay of INSULIN
Introduction
• Insulin was discovered in 1921, by Sir Fredrick bunting which helped millions suffering from type-1
diabetes.
• It is a hormone made in pancreas; special cells call “beta cell” produce insulin. When a person suffer from
type-1 diabetes the capability of these cells is lost. Most people now a days use human insulin or insulin
analogs. It is produced by bacteria (lilly) or by yeast (Novo-Nordisk) by using genetic engineering.
• Human insulin has the molecular weight of 5807 and is made up of two polypeptide chains linked
together by disulfide linkages. 51 Amino acids having 2 chains one is alpha chain = 21 Amino acids and
beta chain = 30 Amino acid
• Insulin preparations are used to control blood – glucose levels in people with diabetes mellitus, which
results from an inadequate secretion of insulin from the pancreas. The insulin preparations used are
mostly human insulin prepared by recombinant DNA or enzymatically modified insulin isolated from
porcine pancreas.
Principle:
• The potency of a sample insulin injection is estimated by comparing the hypoglycemic effect it produces
with that produced by a standard preparation of insulin under conditions of a suitable method of assay.
Mechanism of action
• Every pancreatic islet contains ~1,000 endocrine cells of which 75% are insulin-producing beta-cells.
• Insulin is synthesized as pro-insulin in the endoplasmic reticulum and is processed to the biologically
active form inside the secretory granules.
• The beta-cell is electrically excitable and uses changes in membrane potential to couple variations in
blood glucose to trigger insulin secretion.
• The beta-cell contains about 20 different ion channels proteins
Two types of ion channels are particularly important for the initiation of insulin secretion. The ATP sensitive K+
-
channels are active at low glucose concentrations, because of the high intracellular ADP levels and voltage
sensitive Ca+2
channels
Standard preparation and unit:
• It is pure, dry and crystalline insulin. One unit contains 0.04082 mg. This unit is specified by Ministry of
Health and is equivalent to international unit.
Preparation of standard solution:
• Accurately weigh 20 units of insulin and dissolve it in normal saline. Acidify it with HCl to pH 2.5. Add
0.5% phenol as preservative. Add 1.4% to 1.8% glycerin. Final volume should contain 20 units/ml. Store
the solution in a cool place and use it within six months.
Preparation of test sample solution:
• The solution of the test sample is prepared in the same way as the standard solution.
1. THE RABBIT BLOOD SUGAR METHOD
Selection of rabbits:
They should be healthy, weighing about 1800-3000 gms. They should then be maintained on uniform diet
but are fasted for 18 hrs. before assay. Water is withdrawn during the experiment.

Standard and Sample Dilutions:
These are freshly prepared by diluting with normal NaCl solution so as to contain 1 unit/ml. and 2
units/ml.
USP Reference Standards
1. USP Dextrose RS 2. USP Insulin RS 3. USP Insulin (Beef) RS.
4. USP Insulin Human RS 5. USP Insulin (Pork) RS.
Doses:
The dose which can produce suitable fall in blood sugar level is calculated for the standard.
Principle:
The potency of a test sample is estimated by comparing the hypoglycemic effect of the sample with that
of the std. preparation of insulin. Any other suitable method can also be used.
Experimental Procedure:
• Animals are divided into 4 groups of 3 rabbits each. The rabbits are then put into an animal holder. They
should be handled with care to avoid excitement.
• First part of the Test:
A sample of blood is taken from the marginal ear vein of each rabbit. Presence of reducing sugar is
estimated per 100 ml. of blood by a suitable chemical method. This concentration is called ‘Initial Blood
Sugar Level’.
The four groups of rabbits are then given sc. injections of insulin as follows:
• From each rabbit, a sample of blood is withdrawn up to 5 hrs. at the interval of 1 hr. each. Blood sugar is
determined again. This is known as ‘Final Blood Sugar Level’.
• Second part of the test (Cross over test):
The same animals are used for the second part. The experiment can be carried out after one week. Again,
they are fasted and initial blood sugar is determined. The grouping is reversed, that is to say, those
animals which received the standard are given the test and those which received the test are now given the
standard. Those animals which received the less dose of the standard are given the higher dose of the test
sample and vice-versa. This test is known as „Twin Cross Over Test’. Mean percentage decrease of the
first and second part is calculated by statistical calculations.
2. Mouse Method (Mouse Convulsion Method)
Principle:
 Mice show characteristic convulsions after s.c. inj. of insulin at elevated temperatures. The percentage
convulsions produced by the test and standard preparations are compared.
Experimental procedure:
 Minimum 100 mice weighing between 18-22 gms. of the same strain are used. They should be
maintained on constant diet. They should be fasted 18 hrs. prior to the experiment.

Standard and sample dilutions:
• Dilutions are prepared with sterile saline solution, so as to contain 0.064 units/ml. (std dilution I) and
0.096 units/ml. (std. dilution II). Similarly, test sample solutions are also prepared.
• Mice are divided into 4 groups each containing 25 mice and insulin is injected s.c. as follows
• Mice are put in an air incubator at 33oC and observed for one and a half hr. The mice which convulse or
die are taken out of the incubator and observed. These mice usually convulse severely but failure of the
animal to upright itself when placed on its back, should as well be considered as convulsion. The
convulsed mice may be recovered by 0.5ml of dextrose solution inj.
RESULT
• The result is calculated by standard statistical methods. Refer to Pakistan Pharmacopoeia 1973 for the
details of the processing of the results for the calculation of the potency of the substance being examined.
• The death or convulsions in the test animals is due to the rapid onset of hypoglycemia. The normal blood
sugar levels are 1000 ug/ml. Death or convulsions may occur at any time when the blood sugar conc. falls
below 350 ug/ml
Rat diaphragm method
• Sprague Dawley rats weighing 70–100 g is used. The animals are sacrificed during anesthesia and the
diaphragms still attached to the rib cages are carefully removed, released from the rib cages and adhering
connective and fat tissues, washed in PBS (Phosphate Buffer Saline), spread out and divided into two
equal pieces as described by Müller and coworkers (1994). For assaying the effects of
insulin/compounds/drugs, the hemidiaphragms are incubated in KRH (Krebs ringer HEPES) buffer
gassed with carbogen (95% O2/5% CO2) in the presence of 5 mM glucose
Epididymal fat pad of rats
• Insulin-like activity can be measured by the uptake of glucose into fat
cells. Adipose tissue from the epididymal fat pad of rats has been
found too very suitable.
• The difference of glucose concentration in the medium after
incubation of pieces of epididymal rat adipose tissue or measured
oxygen consumption in Warburg vessels, Radiolabeled 14C glucose,
the 14CO2 is trapped and counted.
The concentration is determined by immuno-assay
I
I
II
II

Bioassay of DIGITALIS
Introduction
Digitalis is Cardio-active glycoside. Acts directly on myocardium and increase force of contraction (used

in CHF)
Principle:
 The activity of a sample is determined by comparing its activity on the cardiac muscle of Guinea pigs or
pigeons with that of standard preparations of Prepared Digitalis to cause death due to cardiac arrest.
Standard preparation and unit
 The standard preparation is the Third international standard, established in 1949 and consisting of dry
powdered leaves of Digitalis Purpurea. It is supplied in ampoules containing approximately 2.5g. The unit
is contained in 76.0 mg of the Standard Preparation; 1mg contains 0.01316 Unit.
Preparation of Extracts
 The contents of a vial containing suitable quantity of the standard preparation are rapidly transferred to a
stoppered weighing bottle and weighed. The contents of weighing bottle are transferred to a hard glass,
glass stoppered bottle of 50ml capacity. Ten ml of alcohol (80%) is added for each gram of powder. The
stopper is inserted and the container shaken continuously for 24 hours at 20 -30 c. The mix is then
immediately centrifuged or filtered through a sintered glass filter with precautions to avoid evaporation of
the solvent. The liquid is transferred to a dry, hard glass bottle, which is tightly closed and kept at a
temperature between 5 c and -5. The extract should be used within one month.
 An extract of substance being examined is prepared in the same manner
Suggested methods
1. By injection into guinea-pigs
2. By injection into pigeons
1. By injection into Guinea-Pigs
 Not fewer than 12 guinea pigs, (200-600g) are distributed at random into two equal groups. (The
weight of heaviest and lightest animal should not differ by more than 100g and the mean body
weight of two groups should not differ by more than 10 %)
 One group is used for the standard preparation and one for the substance being examined.
 The extracts of the std. preparation and of the substance being examined are diluted with saline
solution until the concentrations of ethyl alcohol are the same and do not exceed 10%v/v.
 The diluted extract is injected at a slow uniform rate into the jugular vein of guinea- pig which
has been anaesthetized with urethane and whose respiration may be maintained artificially. The
duration of injection may be between 20 and 40 minutes.
 The injection is continued until the heart is arrested (this may conveniently be determined from
and electrical recording). And the volume of extract administered is taken as the lethal dose. The
mean log lethal dose of (ml) per kg of body weight is determined for each group.
Result: The result of assay is calculated by standard statistical methods. Refer to Pakistan Pharmacopoeia 1973
for the details of the processing of result for the calculation of the potency of the substance being examined.
 When six guinea-pigs are used for standard Preparation and six for the substance being examined, the
fiducial limits of error may be expected to lie between 80-125% of the estimated potency.

2. By Injection into Pigeons
• Not fewer than 12 pigeons are assigned at random to two groups as nearly alike as practicable with
respect to breed, sex and weight. The pigeon should be free from gross evidence of disease, obesity or
emaciation. The weight of the largest pigeon should not exceed twice the weight of the smallest and the
mean weight of the group should not differ by more than 30%.
• One group is used for standard preparation and one for the substance being examined.
• Food but not water is withheld for 16-28 hours before the test.
• The extract of standard preparation and the substance being examined are diluted with saline solution so
that the examined lethal dose per kg of body weight is contained in 15ml and are administered in the
following manner in a sequence determined by randomization procedure.
• The diluted extract is injected through a cannula into the alar vein of the lightly anaesthetized and
immobilized pigeon, using a micro burette.
• The dose is 1ml per kg of body weight and is administered within a few seconds and repeated at five-
minute intervals until the heart is arrested. The amount of extract to produce this effect is taken as lethal
dose.
• If the average number of doses of any given dilution required to produce death is less than 13 or more
than 19 or if the average number of doses of the two groups differ by more than 4, the test is repeated,
using suitable adjusted solutions, freshly prepared.
• The lethal dose (ml) per kg of body weight is equal to the number of doses received. The mean log lethal
dose (ml) per kg of body weight is determined.
Result
• The result of the assay is calculated by standard statistical methods. Refer to Pakistan Pharmacopoeia
1973 for the details of the processing of the results for the calculation of the potency of the substance
being examined.
• When six pigeons are used for Standard Preparations and six for the substance being examined, the
fiducial limits of error may be expected to lie between 80 and 125 % of the estimated accuracy.
• In pigeon, the stoppage of heart is associated with characteristic vomiting response.
• Acceptable 13-19 doses for cardiac arrest or average no. of doses of two groups not differ by more than 4
Potency = mean lethal dose of standard / mean lethal dose of sample

BIOLOGICAL ASSAY OF ANTIBIOTICS , VITAMIN D , DIGOXIN & INSULIN

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