Biological Assays

Introduction:

Assay

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.

Biological Assay or bioassay: (Bio = living, assay = to test)

Biological assay 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 bio-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.

Why bioassay technique is used? (Applications)

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 patient’s sputum helps determine which anti-biotic is given for
quick recovery.
4. Certain complex compounds like Vitamin B-12 which can't be analyzed by
simple assay techniques can be effectively estimated by Bioassays.
5. Chemical assay method for substance is not available or not possible.
6. To measure the drug toxicity.
7. When high sensitivity of drug is required.
8. Bioassay is essential in development of new drug.

The precision, accuracy and reproducibility are depends on proper selection of

tissue or method with highest sensitivity of drug.

Types of bioassay

1. Quantal assay

All or none response in all individuals. E.g. Digitalis induced cardiac arrest in
guinea pigs, hypoglycemic convulsions in mice by insulin.

2. Graded response assay

In these assays, as the dose increase 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 no further increase in response even with an increase in dose.
E.g. Acetyl-choline producing contraction in the muscle of frog Rectus abdominis.

Methods of assays:

There are four methods of assays are used that are classified as

a) Matching point or bracketing method

b) Interpolation assay
c) Three point (2+1) and four point (2+2) assay
a. Matching point or bracketing method

Here a constant dose of the standard is matched 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.

b. Interpolation assay

Here a log dose-response curve is plotted with the standard. The concentration of
the test is then read from the graph.

c. Three point (2+1) and four (2+2) point assay

This method incorporates the principle of interpolation and bracketing. 2+1

indicates- Two 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.

Bioassays 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 antibiotic 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 technique aims to study the biological effect or response of the compound
under screening in a living system directly. Example: 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.

Example: 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.

Preparation of standard
A selective representative sample of a substance for which it is to serve as a basis
of the measurement is called standard preparation.

Types of standard

 International standard and reference standard USP units (highly recognized-

able and authorized standard).
 British standard and reference standard
 Country want to have its own standard preparation, and then used according
to its own law.
 For specific biological activity small quantity of standard preparation are
used.

Potency:

The amount of drug required to produce an effect of given intensity.

For example:

Highly potent drugs like morphine, alprazolam, chlorpromazine etc. produce high
response at low concentration.

Low potent drugs like ibuprofen and acetylsalicylic acid produce low response at
low concentration.

1-Bioassay of antibiotics
The potency of a sample of an antibiotic is determined by comparing the dose
which inhibits the growth of a suitable susceptible micro-organism with the dose of
standard preparation of that antibiotic that produces the same degree of inhibition.

Standard Preparations and Units

The standard preparations are supplied as dry powders in sealed ampoules. The
potency of an antibiotic is usually described 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:
Method:

Petri dishes or rectangular trays are filled to a depth of 3 – 4mm with a nutrient
agar medium which has previously been inoculated with 1 percent v/v of a suitable
inoculums of a susceptible test organism (list of organisms used for particular
antibiotics are given in the table under). The inoculated plates are allowed to dry
for 30 minutes at room temperature or solidified using a refrigerator. Small holes
with diameter between 5 – 8mm are bored in the solidified agar.

Solutions of standard preparations of known concentrations (concentrations used

are given in the following table) are made and solutions of the antibiotic being
tested are made of the same concentrations. The solutions are made with a sterile
solvent and dilutions made unless otherwise stated in the monographs. The
solutions are placed in the holes bored in the agar using a pipette which delivers a
uniform and equal amount of test solution and standard solution in their respective
holes.

The plates are then maintained at room temperature for 4 hours to allow the
antibiotic solutions to diffuse into the agar, the plates are then incubated at a
suitable temperature for approximately 16 hours and the diameters of the zones of
inhibition produced by various concentrations of the test and standard solutions are
measured carefully.

From the results the potency of the test solution is estimated using standard
statistical methods.
2-Bioassay of Insulin Injection:
Insulin is a hormone that is synthesized and secreted by the β-cells of the
pancreatic islets and is stored in intracellular granules. Human insulin has the
molecular weight of 5807 and is made up of two polypeptide chains linked
together by disulfide linkages.

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.

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.

Standard preparation and unit

The standard preparation consists of recrystallized purified insulin. It is supplied in

ampoules containing approximately 110 to 125 mg. One unit is contained in
0.04167 mg of standard preparation.

Preparation of the solution of standard preparation:

A quantity of the standard preparation is dissolved in normal saline such that 1ml
of the solution contains 20 Units.

Method:
Not less than 96 mice which have been well fed are selected and are divided into 4
groups equally at random. The test animals are deprived of food for 2 hours prior
to the test. Two groups receive SC injections of 2 dilutions of the standard
preparation and the other 2 groups receive one dilution each of the sample
preparation having the same dilutions. Suitable doses for mice weighing about 20g
are 0.015 Units and 0.03 Units.

The mice after injection are kept at a uniform temperature between 29 – 35℃ in an
incubator with a transparent front. The mice are observed for 1½ hour after
injection. The number of mice which are dead, convulsed or were lying on their
back for more than 2 – 3 seconds are noted for each group. The results for the
sample preparation are compared with those of the standard preparation and the
assay results are calculated using standard statistical methods.

3-Bioassay of prepared Digitalis

The pharmacodynamic properties of the crude drug extract or galenical preparation
of digitalis cannot be measured adequately by chemical assays because of the
presence of complex mixture of substances containing varying structures and
activity. Hence, biological assay techniques are applied.

The activity of a sample prepared is determined by comparing its activity on the

cardiac muscles of guinea – pig or pigeons with that of a standard preparation of
prepared digitalis.

Standard preparation and unit:

The standard preparation of digitalis consists of dry powdered leaves of Digitalis

purpurea. It is supplied in ampoules containing approximately 2.5g of the standard
digitalis. The Unit is contained in 76mg of that standard preparation and hence, 1
mg contains 0.01316 Units.

Preparation of the extract for standard preparation and sample preparation

The extracts of the standard and test samples are prepared in the same way. First,
the amount of the dry powder is added to a previously weighed stoppered glass
bottle. The amount of the powdered digitalis added is noted. 10 ml of 80% alcohol
is added to the stoppered bottle for each gram of the powder.
The container is continuously shaken for 24 hours at 20 – 30℃ or for 48 hours at
10 – 20℃. The mixture is then centrifuged or filtered through sintered glass filter
taking care to avoid evaporation of the solvent. The extract is stored at a
temperature of -5 to 5℃ and should be used within a month after preparation.

Methods:

By injection into Guinea-pig

Not fewer than 12 guinea pigs each weighing 200 – 600 grams are distributed at
random into two equal groups. The weight of the heaviest and the lightest animals
should not differ more than 100 grams and the mean body weight of the two
groups should not vary more than 10%. One group is used for the standard
preparation and the other is used for the test preparation. Both the test and standard
preparations are diluted with normal saline until the concentration of previously
anesthetized vein of the test animal.

The duration of the injection may be 20 – 40 minutes. The injection is continued

until the heart of the animal is arrested, which can be measured by recording the
electrical activity of the heart. The volume of the extract administered is taken as
the lethal dose. The mean log lethal dose in mL/Kg of body weight is determined
for each group.

The result of the assay is calculated by standard statistical methods.

By Injection into pigeons

Not fewer than 12 healthy pigeons are taken and are divided at random into 2
groups. One group is for the standard preparation and the other for the sample
preparation. The weight of the heaviest pigeon should not me more than twice as
much as the lightest pigeon. Also, the mean weight of both the groups of the
pigeons should not vary more than 30%.

Food, but not water is withheld from the test animals for 16 – 28 hours before the
test. The extracts of both the sample and standard preparation are diluted with
normal saline such that the estimated lethal dose per kilogram body weight of the
pigeon must not exceed more than 15ml. The diluted extract is administered
through a cannula into a previously anesthetized vein of an immobilized pigeon.
The dose of 1ml/kg of the body weight is administered within a few seconds and
repeated after every 5 minutes till the heart of the test animal is arrested.

If the average number of doses received before the heart arrests is less than 13 or
more than 19, or the average number of doses of the 2 groups vary more than four,
the test is repeated using freshly prepared and accurate dilutions.

The lethal dose per kilogram body weight of the pigeon is equal to the dose
administered. The result of the assay is calculated using standard statistical
methods.

4-Bioassay of Vitamin D
The activity of a preparation of vitamin D is determined by comparing its
antirachitic (ability to prevent rickets) activity with that of the antirachitic activity
of a standard preparation.

A fundamental requirement in a microbial assay for the activity of a vitamin is the

inability of the test organism to produce that vitamin. Furthermore, the test
organism must require the factor being assayed for normal growth and should be
sensitive to very small amounts of that factor.

Standard preparation and unit

It consists of activated crystalline 7 – dehydrocholesterol. It is supplied in bottles

as a solution in vegetable oil. One unit is contained in 0.000025 mg of the pure
substance.

Method:

Not fewer than 40 young rats of either sex, recently weaned are chosen and divided
into 4 groups of 10. The weight of the heaviest rat must not vary more than 10
grams of the weight of the lightest rat. They are fed for 3 weeks on a rachitogenic
diet, which consists mostly of carbohydrates, proteins and electrolytes and no fats.
The development of the necessary degree of rickets is determined in each rat under
light anesthesia by the X–ray of the proximal ends of the tibia or the distal ends of
the radius and ulna.
Then the rats in the 2 groups receive dose x and nx of the standard preparation
respectively and the other two groups receive doses in the same ratio of the sample
preparation. Suitable doses may vary from 2 – 8 units, i.e. where x = 2 and n = 1, 2,
3, 4. Each rat may receive the whole of its dose at once or the dose maybe divided
into 8 daily doses. 10 – 14 days after that, the rats are killed and the extent to
which rickets has been cured is estimated by means of X–ray photographs.

The result of the assay of the sample preparation is calculated by comparing it with
the results of the standard preparation by means of standard statistical tests.