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. Both are procedures by which the
potency or the nature of a substance is estimated by studying its effects on living matter. Bioassay is a
procedure for the determination of the concentration of a particular constitution of a mixture.
Bioassays are procedures that can determine the concentration of purity or biological activity of a substance
such as vitamin, hormone, and plant growth factor. While measuring the effect on an organism, tissue cells,
enzymes or the receptor is preparing to be compared to a standard preparation. Bioassays can be classified in
two types:

Qualitative

Quantitative
Qualitative bioassay is used for assessing the physical effects of a substance that may not be quantified, such
as abnormal development or deformity. Example of a qualitative bioassay includes Arnold Adolph Berthold's
famous experiment on castrated chickens. This analysis found that by removing the testes of a chicken, it would
not develop into a rooster because the endocrine signals necessary for this process were not available.
Quantitative bioassays involve estimation of concentration/potency of a substance by measurement of the
biological response it produces. These bioassays are typically analyzed using the methods of biostatistics.
Principles of bioassay
•
Bioassay involves the comparison of the main pharmacological response of the unknown preparation
with that of the standard.
•
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.
•
The test solution and standard should be compared for their established pharmacological effect using a
specified pharmacological technique.
•
The method selected should be reliable, sensitive, and reproducible and should minimize errors due to
biological variation and methodology. (Animals should of same species, sex and weight and number of
animals should be large enough to permit statistical analysis.
Need of Bioassay
1. They not only help to determine the concentration but also the potency of the sample. Potency denotes
activity of the compound 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 used to standardize drugs, vaccines, toxins/ poisons, disinfectants, antiseptics etc. as these are all used
over biological system in some 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 be
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. Sometimes the chemical composition of samples is different but has same biological activity.
6. For samples where no other methods of assays are available.
Procedure
i.
Prepare the physiological salt solution
ii.
Arrange the instrument and adjust the water bath.
iii.
Balance the lever
iv.
Tissue selection
v.
Surgical process and collection of required tissue.
vi.
Tissue attachment to the water bath
vii.
Relaxation time given to the tissue
viii.
Prepare the standard drug( serial dilution)
ix.
Select the lowest possible measurable concentration by trial and error method.
x.
Prepare DRC for the standard drug.
xi.
Prepare DRC for the test drug.( serial dilution)
xii.
Select a assay method (3 point or 4 point assay)
xiii.
Calculation
1. Prepare the physiological salt solution
reagent
Frog-
Kreb’s
Tyrode
Ringer
Ringer-
De
Mc
Locke
Jalon
Ewen
NaCl
65 g
69 g
80 g
91.5 g
90 g
76 g
KCl
1.4 g
3.5 g
2.0 g
4.2 g
4.2 g
4.2 g
MgCl². 6H²O ---
1.1 g
1.0 g
---
---
---
NaH2PO4.
0.1 g
1.4 g
0.5 g
---
---
1.4 g
NaHCO³
2g
21 g
10 g
1.5 g
5g
21 g
CaCl²
1.2 g
2.8 g
2g
2.4 g
0.6 g
2.4 g
Glucose
20 g.
20 g.
10 g.
10 g.
5 g.
20 g
Aerating
air
O²
Pure O²
O² +
O² + 5%
H²O
Gas
+ O² or air
5%CO²
5% CO²
CO²
Frog-Ringer
Kreb’s
Amphibian tissue preparation
Mammalian/Avian skeletal muscle preparation
Tyrode
Intestine preparation
Ringer-Locke
Heart muscle preparation
De Jalon
Rat uterus preparation
2. Arrange the instrument and adjust the water bath.

Student Organ bath
o Outer bath:
First designed by rudolph magnus

Perplex glass

Store water outside the inner bath to maintain the temperature
o Inner bath:
Glass

To observe the tissue during experiment

5-50ml (usually 10ml)
Kymograph: Sherrington- starling kymograph
•
To obtain a graphical amplified measurable response of a muscle or tissue
•
Two important parts: motor box and drum
•
Speed lever: 1 revolution/ 96 min.
•
Paper: glossy side outside – least resistance
Rough side inside – stick to the drum.
•
Fixing solution: shellac and colophony saturated in alcohol
3. Surgical process and collection of required tissue
 Animal sacrificed by cervical dislocation.
 Tissue identified and isolated.
 Carefully dissect and separate unwanted tissue.
 Tissue kept in a physiological salt solution.
 Avoid excessive handling of tissue.
4. Tissue attachment to the water bath
Attach the ends of the tissue: - One end to tissue holder other end to lever
Method of attachment of tissue: Attach the thread at the end by a needle. Care should be taken for
intestine tissue not to block the lumen.
5. Prepare DRC for the standard and test drug
two std doses s1& s2 from linear part of DRC [Let the corresponding response be S1, S2] Also s2/s1 = t2/t1 = 3/2
Types of Bioassays
Basically there are two types of bioassays as per the technique used in determination of the Sample under test.
1. Graded Response Assay
2. End Point or Quantal Assay
Graded Response Assay: In these assays, as the dose increases there are 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 on further increase in response even with an increase in dose.
The graded dose response curve is obtained by plotting a graph with dose on the X-axis and response on the Yaxis. It is usually sigmoid in shape however the log dose response curve is almost a straight line and particularly
useful in bio assay.
Conc. of unknown= Threshold dose of standard/threshold dose of test x Conc. of standard.
Conc. of unknown is read from a standard plot of a log dose response curve of at least 4 sub maximal concentrations
End Point or Quantal Assay
It is the simplest type of the bioassay. The threshold dose of the sample required eliciting a complete or a
particular pharmacological effect is determined and compared with standard.
E.g. Digitalis producing cardiac arrest. Here the sample effect is identified by the response it produces on the
biological system. Digitalis produces cardiac stimulation on further doses it produces cardiac arrest.
(+)d TC (Tubocurarine) producing neck relaxation in rabbit, Here as the sample is injected to the neck muscle
of the Rabbit, the neck starts to droop. On further doses there is complete hanging of the neck and rabbit has no
ability to lift the neck. Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is
done by this method.
•
Based on the method used during the grade point assay procedure for determination of Type of activity and
Potency of the Sample, four methods of assays are classified as:
1. Matching point or bracketing method
2. Interpolation assay
3. Three point (2+1) assay
4. Four- point (2+2) assay
1. Matching point or bracketing method: Here a constant dose of the standard is bracketed by
varying dose of sample until an exact matching between the standard dose responses and the particular dose
response of the sample is achieved.
This technique is used
i.
when test sample is too small
ii.
Inaccurate & margin of error difficult to estimate
iii.
Eg: histamine on guinea pig ileum, Posterior pituitary on rat uterus.
2. Interpolation assay: Bioassays are conducted by determining the amount of preparation of unknown
potency required to produce a definite effect on suitable test animals/organs/Tissue under standard
conditions. This effect is compared with that of a standard. Thus the amount of the test substance required
to produce the same biological effect as a given quantity the unit of a standard preparation is compared and
the potency of the unknown is expressed as a % of that of the standard by employing a simple formula.
3. Multi point Bioassay: 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. It can further divided
as 3 point, 4 point and 6 point bioassay.
•
Three point assay [2+1 dose assay] is Fast & convenient:
•
Log dose response [LDR] curve plotted with varying conc of std drug solutions and given test
solution
•
Select two std doses s1& s2 [ in 2:3 dose ratio] from linear part of LDR [ Let the corresponding
response be S1, S2]
•
Choose a test dose t with a response T between S1 & S2
•
Record 4 sets data as follows

s1
s2
t

t
s1
s2

s2
t
s1

s1
s2
t
Log Potency ratio [M] = [(T –S1) / (S2-S1)] X log (dose ratio)
•
4 point assay [2 +2 dose assay] [E.g. Ach bioassay]
•
Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test
solution
•
Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response be S1,
S2]
•
Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ;
•
Also s2/s1 = t2/t1 = 2/3
•
Record 4 data sets
•
s1
s2
t1
t2
•
s2
t1
t2
s1
•
t1
t2
s1
s2
•
t2
s1
s2
t1
ELISA
The enzyme-linked immuno-sorbent assay (ELISA). An ELISA test uses components of the immune system
and chemicals to detect immune responses in the body. The ELISA test involves an enzyme (a protein that
catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules). ELISA is a
popular format of a "wet-lab" type analytic biochemistry assay that uses a solid-phase enzyme immunoassay
(EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample.
The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control
check in various industries.
 Use of an ELISA test
ELISA tests are widely utilized to detect substances that have antigenic properties, primarily proteins (as
opposed to small molecules and ions such as glucose and potassium). The substances detected by ELISA
tests include hormones, bacterial antigens and antibodies
 Principle of ELISA test
There are variations of the ELISA test, but the most basic type consists of an antibody attached to a solid
surface. This antibody has affinity for the substance of interest, E.g. human chorionic gonadotropin (HCG), the
commonly measured protein which indicates pregnancy. A mixture of purified HCG linked to an enzyme and
the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only
HCG with linked enzyme will bind. The more HCG which is present in the test sample, the less enzyme linked
HCG will bind. The substance the enzyme acts on is then added, and the amount of product measured in some
way, such as a change in color of the solution.
As a wet lab analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the specific substance
whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues
to use liquid reagents during the "analysis" (i.e. controlled sequence of biochemical reactions that will generate
a signal which can be easily quantified and interpreted as a measure of the amount of analyte in the sample) that
stays liquid and remains inside a reaction chamber or well needed to keep the reactants contained; It is opposite
to "dry lab" that can use dry strips - and even if the sample is liquid (e.g. a measured small drop), the final
detection step in "dry" analysis involves reading of a dried strip by methods such as reflectometry and does not
need a reaction containment chamber to prevent spillover or mixing between samples.
Types of ELISA
Four kinds of ELISA here are here illustrated as you may concern:
Direct ELISA
Direct ELISAs involve attachment of the antigen to the solid phase, followed by an enzyme-labeled antibody.
This type of assay generally makes measurement of crude samples difficult, since contaminating proteins
compete for plastic binding sites.
Indirect ELISA
Indirect ELISAs also involve attachment of the antigen to a solid phase, but in this case, the primary antibody is
not labeled. An enzyme-conjugated secondary antibody, directed at the first antibody, is then added. This
format is used most often to detect specific antibodies in sera.
Competitive ELISA
The third type of ELISA is the Competition Assay, which involves the simultaneous addition of 'competing'
antibodies or proteins. The decrease in signal of samples where the second antibody or protein is added gives a
highly specific result.
Sandwich ELISA
The last type of assay is the sandwich ELISA. Sandwich ELISAs involve attachment of a capture antibody to a
solid phase support. Samples containing known or unknown antigen are then added in a matrix or buffer that
will minimize attachment to the solid phase. An enzyme-labeled antibody is then added for detection.
The ELISA method is a benchmark for quantization of pathological antigens and there are indeed many
variations to this method. ELISAs are adaptable to high-throughput screening because results are rapid,
consistent and relatively easy to analyze. The best results have been obtained with the sandwich format,
utilizing highly purified, pre matched capture and detector antibodies. The resulting signal provides data which
is very sensitive and highly specific.
Multiple and portable ELISA
A new technique uses a solid phase made up of an immunosorbent polystyrene rod with eight to 12 protruding
gives. The entire device is immersed in a test tube containing the collected sample and the following steps
(washing, incubation in conjugate and incubation in chromo gens) are carried out by dipping the ogives in micro
wells of standard micro plates filled with reagent.
Applications of ELISA
1. ELISA Test Applications in Antibody Concentration Determination
2. ELISA Test Applications in Monoclonal Antibody Screening
3. ELISA Test Applications in Virus test (HIV, West Nile Virus, NDV)
4. ELISA Test Applications in Home Pregnancy Test
5. ELISA Test Applications in Food industry (detecting potential food allergens such as milk, peanuts,
walnuts, almonds and eggs)
6. ELISA Test can be used to diagnostic diseases
Radioimmunoassay
RIA is a very sensitive in vitro assay technique used to measure concentrations of antigens (E.g. hormone levels
in the blood) by use of antibodies. As such, it can be seen as the inverse of a radio binding assay, which
quantifies an antibody by use of corresponding antigens. Although the RIA technique is extremely sensitive and
extremely specific, requiring specialized equipment, it remains among the least expensive methods to perform
such measurements. It requires special precautions and licensing, since radioactive substances are used. The
unique ability of RIA to measure small molecules can nowadays be achieved in many cases by non-radioactive
methods such as ELISA, where the antigen-antibody reaction is measured using colorimetric, such as
absorbance, fluorescence intensity or polarization.
Principle of radioimmunoassay
It uses an immune reaction [antigen-antibody reaction] to estimate a ligand.
Ag+Ag*+Ab → [Ag -Ab+ Ag*Ab + Ag + Ab*]
- Unbound Ag* and Ag washed out
- Radio activity of bound residue measured.
- Ligand concentration is inversely related to the radio activity.
- [Ag: ligand to be measured; Ag*: radiolabelled ligand].
Method
Requirements:
1. Preparation and characterization of an antigen
2. Radio labeling of the antigen
3. Preparation of the specific antibody
4. Development of assay system.
Components of RIA Assay Kit
1. Drug
2. Antibody
3.
Labeled Drug
To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it
with gamma-radioactive isotopes of iodine, such as 125-I, attached to tyrosine. This radio labeled antigen is
then mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one
another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added.
This causes the unlabeled (or "cold") antigen from the serum to compete with the radio labeled antigen ("hot")
for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody,
displacing the radio labeled variant, and reducing the ratio of antibody-bound radio labeled antigen to free radio
labeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free
antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a standard
curve can then be generated which allows the amount of antigen in the patient's serum to be derived. The
radioactivity for each of a series of known concentrations of standards is used to derive terms for en equation,
often sigmoid curve fit. Once the terms for the equation are derived, the radioactivity is fed into the equation to
obtain concentrations of the unknowns. This function is often integrated into the radioactivity counter, along
with functions indicating coefficient of variation and properties of the binding of tracer to antibody. A typical
RIA will have somewhere around 50% of tracer bound to antibody in a sample with no analyte. Samples are
often run in duplicate to ensure a correct reading.
Applications of RIA
 Endocrinology
 Insulin, HCG, Vasopressin
 Detects Endocrine Disorders
 Physiology of Endocrine Function
 Pharmacology
 Morphine
 Detect Drug Abuse or Drug Poisoning
 Study Drug Kinetics
 Epidemiology
 Hepatitis B
 Clinical Immunology
 Antibodies for Inhalant Allergens
 Allergy Diagnosis
 Oncology
 Carcino embryonic Antigen
 Early Cancer Detection and Diagnosis