Biological Assay
1.
2.
3.
4.
5.
6.
Biological Assays
Biological Assay of Antibiotics
Biological Assays Vitamin D
Biological Assay Insulin Injection
Biological Assay of Digitalis
Biological Assay of d-Tubocurarine
By
H A Rahim
1
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.
• Types Of Assay
 Chemical Assay
 Immunoassay
 Bioassay
2
• Chemical Assay:
It is the study of the separation, identification and
quantification of the chemical components of natural
and artificial materials.
• 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.
• Biological Assay:
Biological testing procedure for estimating the
concentration of a pharmaceutical drug substance in a
formulated drug product or bulk material.
The specific potency of drug is given to animal or
human volunteers and then drug response is compared
3
with the standards.
Comparison of Chemical & Bioassay
Bioassay
Chemical Assay
Less Precise
More Precise
More time consuming
Less time consuming
More expensive
Less expensive
Active constituent &
structure not known
Active constituent &
structure fully established
More sensitive
Less sensitive
More men power
Required
Difficult to handle
Less men power required
Easy to handle
4
Indications 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 variable 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 defined by a chemical
assay e.g. Cis and Trans form of methyl phenidate.
7. Not possible to separate interfering substance e.g.
5
Principles of Bioassay
 All bioassays should be comparative against a
standard drug
 Standard & new drug should be as far as possible
identical to each other
 Activity assayed should be the activity of interest
 The degree of pharmacological response produced
should be reproducible under identical conditions.
e.g. Adrenaline.
 Method of comparison preferably (not essentially)
test therapeutic property of drug.
 Individual variations must be minimized.
6
Classification of Bioassay
There are three types of bioassay:
• Quantal
• Graded
• Effect produced in confined period
• Quantal:
 All or none response in all individuals, e.g. Digitalis
induced cardiac arrest in guinea pigs, hypoglycemic
convulsions in mice by insulin and Calculation of LD50
in mice or rats
 Not précised
 Employed for:
 Comparison of LD50 and ED50
 Comparison of Threshold response
7
Graded Bioassay
 Effect is produced gradually depending on dose
e.g. contraction of smooth muscle preparation
• Accuracy Limits of Bioassay:
“Accuracy improves the efficiency of bioassay
for pharmaceutical biological products.”
An accuracy within 20 % of true value is good.
An accuracy within 10 % of true value is
excellent.
8
Dependency of Biological Testing
Procedure
Biological assay depends on:
 size of the dose
 intensity of drug
It is not necessary that the effect produced in
animals would be same for that produced in
humans. But few exceptions are there that is the
main draw back of bioassay.
9
Partial List of Official Quantitative
Biological Tests
Drug and Dosage form
Test Animal (s)
Antibiotics
Suitable microorganism
Insulin
Rabbit & Mice
Digitalis & other glycosides Pigeon & Guinea Pig
Vitamin A & D
Rat
Parathyroid drugs
Dog
Posterior pituitary
Rat
Tubocurarine Chloride
Rabbit
10
Purpose of Bioassay
1. Compare test sample with standard substance to determine
quantity of test sample required to produce an equivalent
biological response to that of the standard substance.
2. Measuring pharmacological activity of new or chemically
undefined substance.
3. Test method employed in measuring the response of living
animals to toxicity of chemical contaminant. Certain no. of
individuals of sensitive specie are exposed to specific conc.
of contaminant for specific period to examine toxic effects.
4. Investigating function of endogenous mediators.
5. Determine conc. As well as potency of unknown substance.
6. Improving and maintaining standards of basic
environmental conditions affecting well-being of people
e.g. pollutants released by particular source.
7. To determine specificity of compounds to be used e.g.
11
penicillin's are effective against G+ve but not on G-ve.
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.
 Uniform quality
 Stable
Types of Standard Preparation:
• Two type of standard preparation:
1. International standard and reference standard
 USP units (highly recognized-able and authorized
standard)
2. British standard and reference standard
12
• Country want to have its own standard preparation,
then used according to its own law.
• For biological assay and tests, units are referred
called GREAT BRITIAN.
• For specific biological activity small quantity of
standard preparation are used.
13
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
acetyl salicylic acid produced low response at low
conc.
14
Affinity
Efficacy
Advantages
• 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.
15
• 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.
• They not only help to determine the concentration but
also the potency of the sample.
• 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
16
form.
• 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 be given for quick
recovery
• Certain complex compounds like Vitamin B-12 which
can't be analyzed by simple assay techniques can be
effectively estimated by Bioassays.
• Sometimes the chemical composition of samples are
different but have same biological activity.
• For samples where no other methods of assays are
available.
• Biological products like toxin, anti-toxin, sera can be
conveniently assayed. Measure minute (Nano mole & Pico
mole) quantities of active substances can detect active
17
substance without prior extraction or other treatment.
Disadvantages
• Key problem is variability in response
• Large number of animal to be used
• Expertise in experimental design, execution of assay
& analysis of data required
• Leads to expensive & time consuming
• Time related changes ins sensitivity of test organ.
• Tachyphylactic responses of substance being
assayed.
18
BIO ASSAY OF ANTIBIOTICS
&
VITAMIN D
19
For antibiotics assay:
• Introduction
• Preparation of Media
• Preparation of Buffer solution
• Preparation of standard solution
• Preparation of Sample solution
• Methods (cylindrical method & turbidimetric)
For vitamin –D assay:
• Introduction
• Sources
• Stages of assay
• Line test
20
Introduction
• The potency of a sample of an antibiotic is
determined by comparing the dose which inhibit the
growth of micro-organism with the dose of standard
preparation of that antibiotic.
• Two methods used.
• Cylindrical plate method.
• Turbidimetric method.
21
Media
• Prepare the media required for the preparation of test
organism inoculam from the ingredients.
• Dissolve the ingredients in sufficient water to
produce 1000 ml and
• Add sufficient 1 M sodium hydroxide or 1 M
hydrochloric acid, as required so that after
sterilization the pH is as given in monogram. (Table)
Buffer solution:
• Prepare by dissolving the given quantities of
dipotassium hydrogen phosphate and potassium
dihydrogen phosphate in sufficient water to produce
1000 ml after sterilization, adjusting the pH with
phosphoric acid or potassium hydroxide.
22
Quantities in g of ingredients per 1000
ml
23
Table: Buffer Solutions
Prepare by dissolving the given quantities of dipotassium
hydrogen phosphate and potassium dihydrogen
phosphate in sufficient water to produce 1000 ml after
sterilization, adjusting the pH with phosphoric acid or
potassium hydroxide.
Buffer
No.
Dipotassium Hydrogen
Phosphate, K2HPO4 (g)
Potassium Dihydrogen
phosphate, KH2PO4(g)
pH adjusted
after
sterilization to
1
2.0
8.0
6.0±0.1
2
16.73
0.523
8.0±0.1
3
1673
13.61
4.5±0.1
4
20.0
80.0
6.0±0.1
5
35.0
-
10.5±0.1
6
13.6
4.0
7.0±0.2
24
Standard Solution
• To prepare a stock solution, dissolve a quantity of the
Standard Preparation of a given antibiotic, then dilute to
the required concentration as indicated. Store in a
refrigerator and use within the period indicated.
• On the day of assay, prepare from the stock solution five
or more test dilutions, the successive solutions increasing
stepwise in concentration, usually in the ratio 1:1.25 for
Method A or smaller for Method B.
• Use the final diluent specified in monogram.
• Sample Solution:
• Assign & assumed potency per unit weight or volume,
and on this assumption prepare on the day of the assay a
stock solution and test dilution as specified for each
antibiotic in monogram but with the same final diluent as
used for the Standard Preparation.
25
Procedure
• CYLINDER‐PLATE METHOD:
• Inoculate a previously liquified medium appropriate
to the assay quantity of suspension of the micro
organism
• 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.
• Ensure that the layers of medium are uniform in
thickness.
• Using the appropriate buffer solutions, prepare
solutions of known concentrations of the antibiotic to
be examined.
26
• 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
• Incubate them for about 18 hours at the particular
temperature.
• Accurately measure the diameters or areas of the
circular inhibition zones
27
TURBIDIMETRIC/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.
• Prepare five different concentrations of the
standard solution from the stock solution of the
Standard Preparation of the antibiotic &
increasing stepwise in the ratio 4:5.
• Select the median concentration & dilute the
solution of the substance being examined
(unknown) to obtain approximately this
concentration.
28
• Place 1 ml of each concentration of the standard solution
and of the sample solution in each of the tubes in
duplicate.
• To each tube add 9 ml. of nutrient medium
• At the same time prepare three control tubes, one
containing the inoculated culture medium (culture
control), another identical with it but treated immediately
with 0.5 ml of dilute formaldehyde solution (blank) and a
third containing un-inoculated culture medium.
• Place all the tubes, randomly distributed or 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.
29
BIOASSY OF VITAMIN-D
•
•
•
•
•
•
Vitamin-D is a fat soluble vitamin,
Vitamin – D is a sterol, it contains steroid nucleus
Forms of vitamin D:
Vitamin D in the diet occurs in two forms
Vitamin D2 (Ergocalciferol)
Vitamin D3 (Cholecalciferol)
• Sources:
• Butter, Milk, Cheese
• It can be made by the body when the skin is exposed
to ultraviolet light.
• Deficiency of vit-D in children cause rickets disease.
• Excess of vit-D cause irritability, loss of weight,&
30
occasionally death.
• There are three stages of assay:
1: Preliminary period.
2: Assigning rats for assay groups .
3: Assay period.
Preliminary period:
• Not more than 30 days
• Litter of rats are taken in group of 7-8
• Balance diet containing all nutrients except vit-D is given.
• Weight the rats should not <44 & >60gm
• Any physical sign of disease or damage should not be
present
Ingredients
Parts by weight
• Rachitogenic Diet:
Whole yellow corn grounded
76
White glutein grounded
20
CaCO3
3
NaCl
1
31
Assigning rats for assay groups
• Choose rats which should wobbly rachitic gait and joint
become larger than normal
• X-ray examination is done to confirm rickets.
• The rats are divided into four groups.
• To each group one or more assay dose & not <2 dose of
standard should be provided.
• Difference of wt B/w two groups should not be greater
than 8mg.
Assay dose:
• Select two dose level
• Large dose to small dose ratio<1.5 & >2.5
• Duration of group assigning is 30 days
• Dose level of reference & sample is same
32
Assay Period
• The rats in two groups receive standard dose and
other two receive assay (reference) dose.
• 3rd & 4th day assay and standard dose is half
• Environmental condition should be uniform, avoid
anti-ricket radiation (sun light)
• Select rat which don’t show change in weight
• Select rat which consume provided food in 24hrs
• Slaughter these rats and check weather desired level
of calcification is produced
33
Line Test
• Take proximal end of tibia bone & distal end of
radius bone
• Disect & remove tissue attached with them
• Immersed 24hrs in 4% w/v formaldehyde solution
in water
• Cut by longitudinal section and then immersed in
1.5% w/v solution of silver nitrate for a few minutes
and transferred into water
• Exposed to sun light it will cause reaction &
produced desired level calcification
34
Microbiological Assay of Antibiotics
(II)
• Introduction Microbiological assay is a technique in
which the potency or concentration of a compound
is assessed by determining its effect on microorganisms.
• The principles are discussed by Roberts and Boyce.
Microbiological assay is a legal QC requirement for
the assay of a number of antibiotics, in both the
British Pharmacopoeia (BP) and United States
Pharmacopoeia (USP).
• Bioassay compares a reference standard and an
unknown sample, the two preparations being
measured simultaneously.
35
Introduction
• A lot of preliminary work is necessary for microbiological
assays, and if only a small number of samples are
expected irregularly, the method is inefficient. Two assay
methods are normally used, agar diffusion and tube
assays. They have several common features: The
compound being assayed must influence the growth of
the test organism. A varying response in growth must be
produced by addition of varying quantities of the test
material.
• The growth medium must contain an excess of all the
compounds required by the test organism for growth.
The exception to this is the compound being assayed
which should be totally absent from the basic medium.
The assumption is made that the compound being
assayed is the only growth promoting or inhibiting
36
compound present.
Reference standard and units of activity
• The potency (activity) of an antibiotic product is
expressed as the ratio of the dose that inhibits the
growth of a suitable susceptible microorganism to the
dose of an International Biological Standard, an
International Biological Reference Preparation, or an
International Chemical Reference Substance of that
antibiotic that produces similar inhibition.
• Potency of antibiotic can be expressed in “unit” or “μg”
of activity per mg of dried material, as stated in
Pharmacopeia.“
• μg” of activity is based on single active ingredient.
• “unit” is used when there are more than one active
ingredient in the antibiotic.
• 6 Zone of Inhibition
37
Dose response curve
• The dose-response curve is sigmoidal for both
test and standard. The distance between the two
curves (x) measures the effectiveness of the test
material relative to the standard.
• This is the log potency ratio.
• The two lines coincide if standard and test
materials are identical.
38
Large plate assay using Latin square
design
• The Latin Square Design gets its name from the fact
that we can write it as a square with Latin letters to
correspond to the treatments.
• The treatment factor levels are the Latin letters in
the Latin square design.
• The number of rows and columns has to correspond
to the number of treatment levels. So, if we have
four treatments then we would need to have four
rows and four columns in order to create a Latin
square.
• This gives us a design where we have each of the
treatments and in each row and in each column.
39
Large plate assay using Latin square
design
• Latin Square Designs are very efficient designs. It
allow for two blocking factors. In other words, these
designs are used to simultaneously control (or
eliminate) two sources of irritation variability.
Whenever, you have more than one blocking factor a
Latin square design will allow you to remove the
variation for these two sources from the error
variation.
• We can remove the variation from our measured
response in both directions if we consider both rows
and columns as factors in our design.
• 10 Large plate assay using Latin square design
40
Statistical interpretation of microbiological
assay methods
• The assay must be designed in a way that will permit
examination of the validity of the mathematical model on
which the potency equation is based.
• If a parallel-line model is chosen, the 2 log dose-response
(or transformed response) lines of the preparation to be
examined and the reference preparation must be parallel ;
they must be linear over the range of doses used in the
calculation.
• These conditions must be verified by validity tests for a
given probability, usually P = Other mathematical models,
such as the slope ratio model, may be used provided that
proof of validity is demonstrated.
• Unless otherwise stated in the monograph, the confidence
limits (P = 0.95) of the assay for potency are not less than
41
95% and not more than 105% of the estimated potency.
Agar diffusion assay
• These are usually carried out on plates. The assay system is
identical for antibiotics and vitamins. A liquid agar (50C)
preparation of medium minus the compound to be assayed
is inoculated uniformly with a sensitive organism.
• The agar is poured into a petri dish and is allowed to
solidify. The assay material and suitable reference standards
are placed in individual reservoirs.
• These diffuse out into the agar, and the organism grows to
form zones after a suitable period of incubation.
• The zone is one of inhibition (no growth) when antibiotics
or preservatives are used. The zone is one of growth or
exhibition when vitamins or amino acids are used.
• The width of the zone, which depends on the concentration
of active compound used, forms the quantitative basis of the
test.
42
Factors affecting diffusion assay
 Agar nutrient content.
 Cylinder charge volume.
 Thickness (volume) of the agar layer.
 Incubation temperature.
43
Turbidimetric Method
• Reaction tube made from glass or plastic with same
height. Spectrophotometric tube has to be sterile.
• All residues are removed and sterilized before and after
use. 1 ml of test solution and standard solution of each
doses placed in 3 tubes (triple) randomly
• Make 2 control tubes and add 9 ml of inocula into each
tubes
• Place tubes in water bath or incubator at (37)ºC for 2 hrs
• After incubation, add 0.5 ml of dilute formaldehyde
• Measure Transmittance or Absorbance at 530 nm
44
INDUSTRIAL MICROBILOGY
Industrial microbiology is an important area of applied microbiology. It refers to the use of
microorganisms in commercial enterprise and Cheap raw materials are converted to valuable
products through the metabolism of microbes. Microbes for this purpose could be exploited in
different ways.For instance this includes(i) synthesis of fermentation products as acids, alcohols or
other organic compounds(ii) transformation of one compound into another desired type,(iii) the
production of enzymes, antibiotics, or insecticides, or(iv) the use of microbes themselves as food.
20 Pharmaceutical products of microbial origin
There are a number of medicinal substances which derive from microorganisms, including antibiotics,
immunological products and products resulting from recombinant DNA technology.These are
dextran, used as a substitute for plasma, certain enzymes and a small group of organic
chemicals.Chemicals which continue to be produced by fermentation include citric acid, ethanol,
lactic acid, vitamins B2 and B12 and a considerable range of amino acids.
21
Pharmaceutical
products
of
microbial
origin
Various commercial products of economic value made by microbes are (i) medicines i.e.
pharmaceuticals, including antibiotics, steroids, human protein, vaccines, and vitamins, (ii) organic
acids,
(iii)
amino
acids,
(vi)
enzymes,
(v)
alcohols,
22
Pharmaceutical
products
of
microbial
origin
(vi) organic solvents and (vii) synthetic fuels. In addition to these, quite recently potential of microbes
could also be realized in (viii) recovery of metals from ores through bioleaching, (ix) recovery of
petrol, and (x) protein production.
•
23
GMP
in
pharmaceutical
industry
relation
with
Microbiology
The implementation of GMP in the early 1970s, major achievements have been achievement in the
control of microbial contamination in pharmaceutical environment.Microbial contamination of
pharmaceutical products is one of the a major problem for product recall and manufacturing
problems.Knowledge of distribution and survival of microorganism in pharmaceutical environment
is critical in process control of sterile and non-sterile pharmaceutical products.This knowledge is
somewhat limited by the ubiquitous distribution of microorganism in manufacturing facilities, 45
the
diversity of microorganism of pharmaceutical samples, and flexibility of microorganism in surviving
•
•
•
•
24
Microbiologically
controlled
environments
in
the
pharmaceutical
industry
Nonviable particulate and viable microbiological surveillance are used to evaluate the design and
control of a cGMP-manufacturing environment. The nonviable particulate monitoring program
plays an important role as it is used on a routine basis to verify the maintenance of air
classifications.In general, a comprehensive environmental monitoring program should include
scheduled monitoring of -Airborne viable particulatesNonviable particulatesPressure
differentialsDirection of air flowTemperature and humiditySurface microbial contaminants on
personnel and equipment, work tables, floors, and walls.
25 Different methods of microbial monitoring systems for environment in pharmaceutical
industry
Sampling airborne microorganismsPassive MonitoringSettling PlatesActive MonitoringSlit-to-Agar
(STA) Air Sampler (Air through narrow slit, rotational agar plate)Sieve Impactors (Air through a
perforated plate/s)Single-Stage (contact plates or Petri dishes)Multi-Stage Cascade (Stacked
perforated plates)Sterilizable Atrium (Stainless Steel head collection device)Single-Use Sterile
Atrium (Disposable)
26 Different methods of microbial monitoring systems for environment in pharmaceutical
industry
Centrifugal Propeller Sampler (Agar coated strip)Filtration (Polycarbonate, cellulose acetate, gelatin
filters)Impinger (use of liquid medium for particle collection)Real Time Laser-Induced Fluorescence
Systems
27
Sampling
microorganisms
on
surfaces
It is important to perform regular sampling of surfaces within the aseptic processing environment,
including equipment, walls, floors, and counter tops.Contact Plates: Flat surfaces can be sampled
using contact plates. These specialized agar plates are manufactured precisely to ensure a smooth,
evenly distributed layer of agar on each plate. Sampling is achieved by gently rolling the domed
surface of the agar onto the test area. The plate is then incubated under appropriate conditions to
obtain colony counts.Swabs: In an environmental monitoring program, it is important to include the
sampling of surfaces that are not flat or are difficult to access, as these areas may be more difficult to
clean and disinfect. Swabs are preferred for this purpose. Early commercially available swabs
46
presented a lower microbial recovery (30-50%).
Microbial assay of B2 and B12
•
•
•
•
•
•
•
•
•
•
1. MICROBIOLOGICAL ASSAY OF VITAMIN B2 AND B12 BY PANKHIL GANDHI, S.Y. PHARM D.
2. WHAT IS MICROBIAL ASSAY OF VITAMINS?
3. • Microbiological 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 their growth are standardized by microbial assay.
4. WHAT IS THE UNDERLYING PRINCIPLE? • The basis of this assay is to measure the ability of test
organism to utilize the substance being assayed under a proper nutritional condition. • The response
(growth of test organism) is proportional to the dose (amount of factor) added to medium.
5. HOW DO WE DO IT ? The Indian pharmacopoeia gives two methods: • The cylinder-plate (cupplate) method • The turbidimetric (tube assay) method
6. ASSAY OF VITAMIN B12
7. A QUICK REVIEW OF VIT. B12 • Also known as cyanocobalamin. It’s a water soluble vitamin. •
Structure is similar to that of heme where the iron is replaced with cobalt as a centre of molecule. • Its
main sources are liver, eggs, milk, meat & fish. • VitB12 deficiency causes Macrolytic anemia,
pernicious anemia. • National Research Council, USA recommends a daily intake of about 5mg of vit
B12
8. PREREQUISITES FOR THE ASSAY: • Test organism selected must be able to utilize free
cyanocobalamin • LACTOBACILLUS LEICHMANNII satisfies this requirement • It is easily available,
non pathogenic and easily culturable • Isolated from milk, cheese and other dairy products
9. PRECAUTIONS • Great care must be taken to avoid contamination • All the glasswares must be free
from detergents and other chemicals. • Glasswares must be heated to 2500C for at least 1 hr before use.
• The whole experiment must be carried out under proper aseptic condition
10. REAGENTS • STANDARD B12 STOCK SOLUTION A solution of cyanocobalamin of concentration
1.0 microgram per ml is made using 25% ethanol • STANDARD B12 SOLUTION dilute stock solution
to prepare a solution of conc. 0.01-0.04 microgram/ml . Prepare freshly
47
•
•
•
•
•
•
•
•
•
•
11. • TEST SOLUTION TO BE ASSAYED Accurate amount of material to be assayed is taken &
dissolved in water, Dil HCl or NaOH is added to adjust ph at 6.0.
12. • BASAL MEDIUM STOCK SOLUTION DISSOLVE IN 100 ml WATER
13. • SUSPENSION MEDIUM Prepare a 100ml solution by mixing equal volumes of BASAL
MEDIUM STOCK SOLUTION and distilled water
14. • PREPARATION OF INOCULUM 1. Transfer a loopfull of Lactobacillus liechmannii from a
recent subculture into two tubes each containing 10ml of sterile culture medium. 2. Incubate
the tubes for 18-24hrs @ 37oC. 3. Centrifuge. Make a suspension, of the cells that settle down,
using 10ml of sterile suspension medium. 4. Centrifuge again and once again suspend the cells
in 10 ml sterile suspension medium. 5. Aseptically transfer 1ml of the so prepared suspension to
10ml sterile suspension medium. 6. This suspension is used as innoculum.
15. COMPOSITION OF CULTURE MEDIUM : (PH = 6.8) • Yeast extract -0.75gm • Peptone -0.75gm
• Dextrose -1gm • Potassium dihydrogen phosphate -0.2gm • Tomato juice filtrate -10ml •
Sorbitan monooleate solution-1ml • Water upto-100ml
16. WHAT IS THE PROCEDURE? Assay of vitamin B12 can be carried out by two methods 1.
Titrimetric method 2. Turbidimetric method
17. TITRIMETRIC METHOD
18. INTERPRETATION OF THE RESULTS
19. OVERVIEW
20. TURBIDIMETRIC METHOD • Incubate all the test tubes at 30-37oC for 16- 24hrs • Set the
48
wavelength of the specrtophotometer at 640nm • Take readings
•
•
•
•
•
•
•
•
•
•
•
•
•
•
21. ASSAY OF VITAMIN B2
22. A QUICK REVIEW OF VITAMIN B2 • Also known as riboflavin • A component of coenzymes FAD and FMN.
Therefore plays important role in carbohydrate and protein metabolism • Dietary sources include yeast, liver, beef, eggs,
whole-grain products, peas, beet, peanuts, etc • Deficiency may lead to blurred vision, dermatitis and anemia.
23. PREREQUISITES • LACTOBACILLUS CASEI is used for the assay • Basal medium • Stock solutions • Inoculum
24. BASAL MEDIUM (RIBOFLAVIN FREE)
25. RIBOFLAVIN SOLUTIONS
26. INOCULUM • Into a 10 ml centrifuge tube add 5 ml basal medium and 5 ml riboflavin solution (0.2 microgram per ml)
so that it would contain 1 microgram of riboflavin • Sterilize • Add LACTOBACILLUS CASAEI to it • Incubate at 37oC for
24 hrs • Centrifuge it and decant off the supernatant liquid
27. • Suspend the bacterial cells in 10 ml sterile saline • Centrifuge again and suspend the bacterial cells in 10 ml sterile
saline • Inoculum is ready
28. PROCEDURE • Fill 7 test tubes with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ml of standard riboflavin solution • Make up the volume
in each test tube up to 5 ml using distilled water • Add 5 ml of riboflavin free basal medium in each of he test tubes •
Sterilize
29. • Take another 5 test tubes and fill them with 0.5, 1.0, 1.5, 2.0, 2.5 ml of the test sample • Make up the volume in each test
tube up to 5 ml using distilled water • Add 5 ml of riboflavin free basal medium in each of he test tubes • Sterilize
30. • Cool all the test tubes to room temperature and then inoculate each test tube with a drop (0.6 ml) of inoculum •
Incubate at 37oC for 72 hrs • Titrate the contents of each test tube separately against 0.1N NaOH to pH 6.8
31. OVERVIEW
32. ALTERNATIVELY.... • Even LACTOBACILLUS RHAMNOSUS is used • Only things that change are composition of
basal medium and concentration of riboflavin solution used • The most effective assay range is between 0.025-0.15
microgram per 10 ml
33. BASAL MEDIUM
34. REFERENCES 1. BISHNOI KAPIL et al, MICROBIOLOGICAL ASSAY FOR VITAMIN B, INTERNATIONAL
RESEARCH JOURNAL FOR PHARMACY, 2012, PG NO 74-82 2. A. A. HOOVER AND G. C. N. JAYASURIYA,
MICROBIOLOGICAL ASSAY OF VITAMINS 2 – RIBOFLAVIN, CEYLON J. MED. SCI. (D), VOL 8, PART 3, FEBRUARY 49
1953, PG NO 184-189 3. DIFCO AND BBL MANUAL, 2ND EDITION 4. INDIAN PHARMACOPOEIA – 2007, VOLUME-1,
BIO-ASSAYS OF INSULIN
INDEX
• INTRODUCTION
• MECHANISM OF ACTION
• ASSAY OF INSULIN
– preparation of Standard solution
– Preparation of test sample solution ›
•
•
•
•
Rabbit Method
Mouse Method
Rat diaphragm method
Rat epididymal fat-pad method
50
Introduction
• Insulin was discovered in 1921, 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..
• Its is produced by bacteria (Lilly) or by yeast
(Novo-Nordisk) by using genetic engineering….
51
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 KATPchannels are active at low glucose concentrations,
52
because of the high intracellular ADP levels.
Mechanism of action of insulin
53
Bioassay of insulin
• 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, Government
of India 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.
54
Rabbit method
• Selection of rabbits: They should be healthy,
weighing about 1800-3000 gm. 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.
• 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.
55
• 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:
12 Rabbits
3
Std. dilution
(I)
3
Std. dilution
(II)
3
Test sample
dilution (I)
3
Test sample
dilution (II)
56
• 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 viceversa. This test is known as ‘Twin Cross Over Test’.
57
Mouse Method
• 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 gm. 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.
58
• Mice are divided into 4 groups each containing
25 mice and insulin is injected s.c. as follows ›
25
Std. dilution
0.064units/
ml
25
Std. dilution
0.096units/
ml
100
Mice
25
Test sample
dilution
25
Test sample
dilution
• 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
59
convulsion.
Rat diaphragm method
• Sprague Dawley rats weighing 70–100 g are 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, 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 buffer
gassed with carbogen (95% O2/5% CO2) in the
presence of 5 mM glucose
60
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 to 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, Radio labeled
14C glucose, the 14CO2 is trapped and counted.
• The concentration is determined by immunoassay.
61
Bioassay of Digitalis, d-tubocurarine ,
Oxytocin
62
Bioassay of Digitalis
PRINCIPLE:
• The potency of test sample is determined by comparing its
activity on cardiac muscle with that of standard preparation
of digitalis under the conditions of suitable method of
bioassay.
STANDARD PREPARATION: 1Unit = 76mg
• It consists of mixture of dried & powdered digitalis leaves
PREPARATION OF TEST EXTRACT:
• Extracting Digitalis powder with Dehydrated alcohol in a
continuous extraction apparatus for 6 hours. The final
extract should contain 10ml (5ml Water+ 5ml Alcohol) per
10gm of digitalis powder.
ASSAY METHODS:
• Guinea pig method
• Cat method
• Pigeon method
63
Guinea pig Method
• Test and standard extracts are diluted with normal saline
in 1:80 ratio(i.e.; 1g extract in 80ml Saline).
• A guinea pig is anaesthetized with suitable anaesthetic.
• It is then dissected to cannulate its Jugular vein using
venous cannula. A pin is inserted in to apex of the heart.
• The standard preparation is injected through the venous
cannula until the heart stops. Amount of extract utilized
to produce this effect is taken as Lethal dose.
• Another 19animals are used to determine mean Lethal
dose.
• Mean lethal dose of test sample is then determined in a
similar manner using atleast 6 guinea pigs of same strain.
• The potency of unknown substance is calculated as ‘
Concentration
= Mean lethal dose of std X Conc. Of sample/ Mean lethal
dose of sample x Conc. of std.
64
Cat Method
• A cat is anaesthetized using Chloralose & its BP
is noted.
• The standard preparation is slowly infused in to
the animal until cardiac arrest occurs & the
blood pressure falls to zero.
• The amount of extract required to produce this
effect is taken as Threshold dose.
• The procedure is repeated using test extract &
then the potency of test sample relative to
standard is calculated.
65
Pigeon Method
• A Minimum of 6 disease free pigeons are used for
testing each sample.
• They are fasted for 16-28hrs before the experiment.
• They are anaesthetized with anaesthetic ether to
dissect one side if their wings & to cannulate their
Alar vein through venous cannula.
• The standard preparation is injected through the
cannula until the heart stops & emesis is produced.
• The volume of fluid extract required to produce
emesis is take as Lethal dose.
• The mean lethal dose of both standard & test
preparation is determined & the potency of test
66
sample relative to standard is calculated.
Bioassay of d-Tubocurarine
• Two methods are used:
1. Rabbit Head-drop Method:
2. Frog Rectus Abdominis muscle preparation
67
Rabbit Head-drop Method:
PRINCIPLE:
• d-tb is an Non-depolarizing neuromuscular
blocking drug which mainly acts as an antagonist
at cholinergic receptors of motor end plate. Hence,
it blocks neuromuscular transmission &causes
MOTOR PARALYSIS.
• It is injected into the marginal ear vein of a rabbit’s
ear until the neck muscles are paralyzed & rabbit
drops its head.
• The total amount of test sample required to
produce head drop is compared with that of the
standard preparation.
68
Procedure:
• Healthy rabbits weighing 2kgs are selected.
• A minimum of 4 rabbits are used for testing each
sample.
• Each rabbit is placed in a holder with its head
protruding outside, such that the head is freely
movable.
• Standard preparation of d-tb (0.012% w/v saline) is
infused in to the marginal vein at a rate of 0.4ml/min
until the head drop s seen.
• The mean dose of standard preparation that produces
head drop is determined.
• Mean dose of test sample which produces head drop
in rabbits is similarly determined & compared with
69
that of standard.
Precautions:
• Neostigmine methyl sulphate(0.05mg) & Atropine
sulphate should be immediately injected in to the
marginal vein to avoid Respiratory distress.
• Cross-over test [Rabbits which receive std
preparation on 1st day should be treated with test
sample on 2nd day & vice versa] should be carried
to minimize biological error.
70
Frog Rectus Abdominis muscle preparation
PRINCIPLE:
• d-tb is a neuromuscular blocking agent which
antagonizes the actions of Ach by blocking nicotinic
cholinergic receptors in the skeletal muscles. Hence, the
actions of Ach are inhibited in the presence of d-tb.
• The CRC of Ach shifts to the right in the presence of d-tb.
PROCEDURE:
• A frog is pithed & laid on its back on a dissecting board
to which it is pinned.
• The skin covering the abdomen is removed to expose the
rectus abdominis muscle.
• Rectus abdominis muscle is then cut, tied with a thread
at its two ends & suspended in organ bath containing
Ringer solution under a tension of 1g.
71
• The tissue is oxygenated to keep it alive & allowed
to relax for 30-45 mins during which the tissue is
washed with a fresh quantum of ringer solution
atleast thrice.
• Contractions due to Ach using atleast 4 increasing
doses are recorded using Frontal writing lever.
• d-tb(1-2 u g/ml) is added to the reservoir containing
ringer solution & allowed to act for 30mins.
• The CRC of Ach I the presence of d-tb is repeated.
• The % inhibition in the response of Ach due to d-tb
is calculated & a log DRC for standard drug is
plotted.
• The potency of the test sample is calculated from the
standard curve.
72
73
Bioassay of Oxytocin
• Depression of BP in Chicken
• Contraction of Rat uterus
• Measurement of Milk-ejection pressure in a
lactating rat
74
Principle:
• Potency of the test sample is compared with that of
the standard preparation.
Standard preparation:
• The standard preparation is the 4th International
standard for oxytocin established I 1978, consisting
of Freeze-dried synthetic oxytocin peptide with
human albumin & citric acid or any other suitable
preparation, the potency of which has been
determined in relation to the International standard.
75
Procedure:
Depression of BP in chicken:
• A young healthy adult cockerel weighing 1.2-2.3 kg is
selected & anaesthetized with a suitable anaesthetic.
• The gluteus primus muscle of one thigh is cut &
retracted to expose the popliteal artery & cural
{Brachial} vein.
• The popliteal artery & cural vein are cannulated.
• The standard preparation is diluted with saline before
use & then two doses of preparation are injected into
the vein through the cannula at an interval of 3-10
mins.
• The decrease in Bp is noted.
76
• The test sample is also diluted with saline to
obtain response similar to that of standard &
two test doses are selected such that the ratio
b/w them is same as the ratio between two std
doses.
• The test doses are injected in to the cannulated
vein & the decrease BP is recorded.
• A minimum of 6 responses are measured for
each sample & calculated using standard
statistical methods.
77
Contraction of Rat uterus:
• Female rats weighing 120-200g are selected.
• About 18-24hrs before the assay, 100ug of Oestradiol
benzoate is injected i.m. to attain oestrous/
proestrous stage.
• The rat is sacrificed & one horn of uterus is isolated
& suspended in an organ bath containing solution
with the following composition:
Components
Composition (% w/v)
NaCl
0.663
KCl
0.045
CaCl2
0.007
NaHCO3
0.256
NaH2PO4
0.003
MgCl2
0.010
C6H12O6 (Dextrose)
0.050
Na2HPO4
0.029
78
• The bath is maintained at a temperature of 32ºC.
• The tissue is oxygenated to keep alive &allowed to
relax for 30min under a tension of less than 2g.
• Two doses of standard preparation suitably diluted
with the above solution are added at an interval of 3-5
mins.
• The contractions produced by the addition of two
standard doses are recorded.
• The test sample is also suitably diluted with above
solution & two test doses with the same interdose ratio
are selected, added,& then contractive doses are
recorded.
• A minimum of 6 responses are measured for each
sample & calculated using standard statistical methods.
79
Measurement of Milk-ejection
pressure in lactating Rat:
• A lactating rat in 3-21 days after parturition & weighing
about 300g are selected.
• It is separated form the litter & 30-60mins later, it is
anaesthetized with phenobarbitone sodium.
• It is then toed to the operating table maintained at 37oC
by its hind legs leaving the front legs free.
• The trachea is cannulated with a short polyethylene tube
with an internal diameter of 2.5mm to facilitate artificial
respiration.
• An external jugular/femoral vein is then cannulated
with a polyethylene tube of internal diameter of 0.4mm.
80
• The skin surrounding the inguinal & abdominal
teats is shaved & tip of one of the teat is excised.
• A polyethylene tube of internal diametre0.3mm&
external diameter 0.6mm is inserted into the
primary teat duct which opens onto the cut
surface & tied firmly in place with a ligature.
• This tube is connected to a suitable strain gauge
transducer.
• The whole system is the filled with 3.8% w/v
sodium citrate or saline solution containing
50units of Heparin per ml to prevent milk clotting.
• The strain gauge is clamped to apply a slight
tension to the teat & to preserve its natural
alignment.
81
• The gauge is connected to a potentiometric recorder
which gives a full scale deflection for an increase in
milk ejection pressure of about 5.3kpa.
• Standard & test samples are being examined are
prepared in saline solution.
• Two doses of standard preparation are selected such
that an increase in milk-ejection pressure is about
1.35kpa for the lower dose & about 2.7kpa for the
higher dose.
• The two doses are injected into the cannula & the milk
ejection pressure are recorded. Two doses of test
preparation with same inter-dose ratio are selected,
injected in to the cannula & response is recorded.
• A minimum of 4responses of each preparation are
measured & calculated by standard statistical methods.
82