ESAI BIOLOGIS

BIOLIGICAL ASSAYS
Disain dan Analisis Bioesei

Urip Harahap

PROGRAM MAGISTER ILMU FARMASI

FAKULTAS FARMASI USU
2020
TTM-1
 OBJEKTIF:
Setelah mengikuti kuliah ini mahasiswa mampu:
Menjelaskan prinsip dasar esei biologis
Mendiskusikan keuntungan relatif esei biologis dibandingkan
dengan metoda fisika dan kimia
Menggambarkan problema utama jika dikaitkan dengan
penggunaan hewan atau jaringan hewan dalam esei biologi
Menjelaskan standar yang digunakan dalam esei biologi dalam
term yang umum
Menggambarkan makna aktivitas biologi
Menjelaskan efek yang diperoleh dalam esei biologi pada hewan
atau jaringan
Mendiskusikan aspek disain eksperimen esei biologi
Memahami teknik-teknik esei biologis baik dalam menguji bahan
alam yang belum pernah diketahui kahsiatnya secara farmakologis
(kualitatif) dan bahan atau obat yang telah ditehui aktivitas
biologinya (kuantitatif).
 Principles OF
Bioassay

Agen: Mengukur dan membandingkan

Estimasi atau Fisik besar respons bahan uji dengan
Determinasi Kimia standar pada sistem biologis
Biologis sesuai dengan kondisi standar

Metode kimia: metode yang sangat
kompleks, memerlukan dosis kimia
tinggi dalam ujinya dan komposisi kimia
tersebut yang menunjukkan tindakan
farmakologis
Bioassay: instrumen yang dapat digunakan
untuk mengestimasi dan penemuan zat aktif
biologis dan aplikasinya penting karena
secara farmakologi memiliki sensitivitas dan
memeiliki spesifisifitas
 What is Bioassay?
• A bioassay is the use of a living organism to
test for the presence of a compound or to
determine the amount of the compound that is
present in a sample.
• The organism used is sensitive to the compound
for which the test is conducted. Thus, the effect
observed is typically the death or deteriorated
health of the test organism. Depending on the
test organism, soil, air, or liquid samples can be
assayed.
 What is Bioassay?
• The classic historical example of a bioassay was the
use of canaries by miners in past centuries. Because
canaries are more sensitive than humans to noxious
gases like methane, they reacted quickly to even small
amounts of the gas. This would give the miners time to
escape.
• Some species of plants can also be used to accomplish
bioassays in the water. More commonly, however, the
test organisms are single-celled organisms such as
algae, water fleas (in particular a species
called Daphnia magna, or fish (in particular the fathead
minnow).
 BIOASSAY
Bioassay is a shorthand commonly used term for
biological assay and is a type of scientific experiment.
Bioassays are typically conducted to measure the effects
of a substance on a living organism. Bioassays may be
qualitative or quantitative. Qualitative bioassays are used
for assessing the physical effects of a substance that may
not be quantified, such as abnormal development or
deformity. Quantitative bioassays involve estimation of
the concentration or potency of a substance by
measurement of the biological response that it produces.
Quantitative bioassays are typically analyzed using the
methods of biostatistics
• Bioassay kata yang umum digunakan untuk terma
biological assay dan merupakan jenis eksperimen ilmiah.
Bioassays adalah jenis eksperimen yang dilakukan untuk
mengukur efek suatu substans pada organisme hidup .
• Bioassays bisa dilakukan secara qualitative atau
quantitative.
• Qualitative bioassays : digunakan untuk menilai efek fisik
suatu senyawa yang tidak bisa dikuantifikasi, seperti
abnormal development or deformity.
• Quantitative bioassays mengestimasi kadar suatu senyawa
dengan cara mengukur respons biologi yang dihasilkannya.
Quantitative bioassays dianalisis menggunakan metode
biostatistics
Metode untuk mengukur efek obat sangat diperlukan
agar kita dapat membandingkan sifat-sifat zat yang
berbeda, atau substans yang sama pada circumstance
yang berbeda, persyaratan ini dipenuhi oleh teknik
bioassay, yang didefinisikan sebagai estimasi
konsentrasi atau potensi suatu bahan dengan cara
megukur respons biologisnya pada living systems
Bioassays are essential in the development of new drugs,
and monitoring pollutants in the environment.
Environmental bioassays are generally a broad-range
survey of toxicity, and a toxicity identification evaluation
is conducted to determine what the relevant toxicants are.
The use of bioassays include:
1. measurement of the pharmacological activity of new or
chemically undefined substances
2. investigation of the function of endogenous mediators
3. determination of the side-effect profile, including the degree of
drug toxicity
4. measurement of the concentration of known substances
(alternatives to the use of whole animals have made this use
obsolete)
5. Assessing the amount of pollutants being released by a particular
source, such as wastewater or urban runoff.
 What is Bioassay?

• Today's bioassays are more sophisticated than the

canary. The ASTM (formerly known as the American
Society for the Testing of Materials) has catalogued
over 70 different bioassays. These are used to analyze
soil, freshwater, and the sediment at the bottom of
watercourses like streams and rivers, saltwater, and
air.
 What is Bioassay?
• Plants can be used as indicators of the presence of
toxic compounds in the soil. In this bioassay, seeds
or the mature plant is introduced into the soil of a
site that is suspected of being contaminated.
• Failure of the seeds to germinate, or failure of the
mature plant to thrive, can be evidence of
contamination. If the assay is done in a controlled
manner with the use of standards to provide
reference points, then the geographical area of
contamination can be determined.
 What is Bioassay?
Bioassay or biological assay/screening is any
qualitative or quantitative analysis of a
substances that uses a living system, such as an
intact cell, as a component.
 Application of bioassay in pharmacology

• Determination of drugs potency

• Determine the therapeutic advantage of one
drug over anther treatments
• Screening of new agents
• Determination of the pharmacological
activities of a new drug
• Establishment of SAR
 Comparison between Bioassay and
Chemical assay
• Bioassay is less precise
• In bioassay, active constituent and its
structure do not have to be known
• Bioassay is more sensitive
 TYPES OF ASSAYS
• Chemical Assays: Spectrophotometry,
Spectrofluorimetry, Chromatography,
• Immunoassays
• Microbiological assays
 Biologicals Substances which
cannot be completely
characterised by physicochemical
techniques alone, and which
therefore require the use of some
form of bioassay.
ƒ
 SUBJECT
• Bacteria can be used in bioassays. For
example, the use of bacteria to detect and
determine the amount of antibiotics or
compounds that might be carcinogens in a
sample has been practiced for decades.
 SUBJECT
• Another particularly useful bacterial bioassay
involves the use of bacteria that have been designed
to fluoresce (to emit light). If the bacteria are
harmed by a toxic compound in the test sample,
then they fail to fluoresce. The decrease in
fluorescence of bacterial populations is measured
in a device called a spectrophotometer. The degree
of decrease can be compared to standards in order
to determine the concentration of the toxic
compound.
 QIUALITATIVE AND QUANTITATIVE
BIOSSAY
• 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
 ESSENTIAL COMPONENTS OF
BIOASSAYS/ASSAYS

Stimulus (Test sample, drug candidate, potential

agrochemical, etc)
Subject (Animal, Tissues, Cells, Sub-cellular
orgenlles, Biochemicals, etc.)
Response (Response of the subject to various
doses of stimulus)
 Bioassays/Assays

 Whole animals
 Isolated organs of vertebrates
 Lower organisms e.g. fungi, bacteria, insects,
molluscs, lower plants, etc.
 Cultured cells such as cancer cells and tissues
of human or animal organs
 Isolated sub-cellular systems, such as enzymes,
receptors, etc
 WHY WE NEED TO PERFORM
BIOASSAY?
 Untuk memprediksi beberapa jenis potensi terapi, baik
secara langsung atau dengan analogi dari senyawa uji.
 Bioassay: istilah yang umum digunakan untuk uji
biologis dan biasanya jenis percobaan in vitro
 Bioassay biasanya dilakukan untuk mengukur efek
senyawa (zat) pada organisme hidup atau sampel hidup
lainnya.
 Types of Bioassays?
 In Silico Screenings
 Non- physiological Assays
 Biochemical or Mechanisms-Based Assays
 In Vitro Assays
 Assays on Sub-cellular Organelles
 Cell based Bioassays
 Ex-Vivo Assays
 Tissue based Bioassays
 NMR Based Drug Discovery
 In Vivo Bioassays
 Animal-based Assays/Preclinical Studies
 Human trial/Clinical Trials
 The Bioesei Basic Principles and the types
of bioesei effects
• Bioassay is defined as estimation or determination of
concentration or potency of physical, chemical or biological
agents 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.
• Bioassay is a successful tool in estimation and discovery of
biologically active substances and important application in
sensitivity and specificity of pharmacological applications.
• Chemical method is very complex method which requires
high chemical dosage and chemical composition shows the
pharmacological actions against the drug.
 IMPORTANCE OF STANDARDS IN
BIOASSAYS/ASSAYS

• The results of the assay/bioassay need to validated by

monitoring the effect of an available known
compound (Standard).
• Without judicious choice of standard and its
reproducible results in an assay system, no screening
can be claimed credible.
 IN VITRO BIOASSAYS

 In Vitro: In experimental situation outside the

organisms. Biological or chemical work done
in the test tube( in vitro is Latin for “in glass”)
rather than in living systems
 Examples include antifungal, antibacterial,
organ-based assays, cellular assays, etc
 Examples of In Vitro Bioassays

 Activity Assays
DPPH assay
Xanthine oxidase inhibition assays
Superoxide scavenging assay
Antiglycation assay
 Bioassays (cell-based)
DNA Level
Protein Level
RNA Level
Immunology assay
 Toxicity Assays
MTT assay
Cancer cell line assays
 IN VIVO SCREENINGS OR
PHARMACOLOGICAL SCREENINGS

 In Vivo: Test performed in a living system such

as antidiabetic assays, CNS assays,
antihypertensive assays, etc.
 Examples of In Vivo Bioassays
 Animal Toxicity
Acute toxicity
Chronic toxicity
 Animals Study
Animal model with induced disease
Animal model with induced injury
 Pre-Clinical Trials
 Clinical Trials
 INDICATIONS FOR BIOASSAY
• Active principle of drug is unknown
• Active pple cannot be isolated, e.g. insulin, posterior
pituitary extract etc.
• Chemical method is either
– not available
– if available, too complex,
– insensitive to low doses e.g. Histamine can be bioassayed in
microgram conc.
• Unknown Chemical composition, e.g. long acting thyroid
stimulator.
• Chemical composition of drug variable but has same
pharmacological action e.g. cardiac glycosides isolated
from diff sources, catecholamines etc.
 USES OF BIOASSAY
• The uses of bioassay are:
 To measure the pharmacological activity of new or
chemically undefined substances to investigate the
function of endogenous mediators
 To measure drug toxicity and unwanted effects.

Bioassays are critical for:

 Product development and manufacturing
 Ensuring continued quality, safety and efficacy of
biopharmaceutical products
 Bioassay plays a key role in the development of new drugs
 Principle of Bioassays
To compare the test
substance with the
international
standard preparation
of the same and to
find out how much
test substance is
required to produce
the same biological
effect, as produced
by the standard
 In Vitro Techniques
 These techniques emplos cell
culture of recommended
biological system to studythe
effectof compound under
standard condition not similar
to that of living
environtment.Here the cell
culture sruvives by utilization
of the nutrition in the media `
 Example: use ofstemcells, cell
culture, microbes (bacteria, etc
 In Vivo Techniques
 These techniques
employs a living
animal recommende
for the purpose of
assay. The techniques
aims tostudythe
biological effect or
response of the
compound under
screening in a living
system directly
 Example: bu use of
mice, rabiit, dog, etc
Characteristics ofgood assays methode
 REFERENCE
1. https://www.bioassay.de/biological-assays/
2. https://www.sciencedirect.com/topics/agricult
ural-and-biological-sciences/bioassays
3. https://www.omicsonline.org/bioassay-peer-re
viewed-open-access-journals.php
4. RRJOB | Volume 3 | Issue 2 | April - June,
2015