Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
How People Really Hold and Touch (their Phones)Steven Hoober
For the newest version of this presentation, always go to: 4ourth.com/tppt
For the latest video version, see: 4ourth.com/tvid
Presented at ConveyUX in Seattle, 7 Feb 2014
For the newest version of this presentation, always go to: 4ourth.com/tppt
For the latest video version, see: 4ourth.com/tvid
We are finally starting to think about how touchscreen devices really work, and design proper sized targets, think about touch as different from mouse selection, and to create common gesture libraries.
But despite this we still forget the user. Fingers and thumbs take up space, and cover the screen. Corners of screens have different accuracy than the center. It's time to re-evaluate what we think we know.
Steven reviews his ongoing research into how people actually interact with mobile devices, presents some new ideas on how we can design to avoid errors and take advantage of this new knowledge, and leaves you with 10 (relatively) simple steps to improve your touchscreen designs tomorrow.
What 33 Successful Entrepreneurs Learned From FailureReferralCandy
Entrepreneurs encounter failure often. Successful entrepreneurs overcome failure and emerge wiser. We've taken 33 lessons about failure from Brian Honigman's article "33 Entrepreneurs Share Their Biggest Lessons Learned from Failure", illustrated them with statistics and a little story about entrepreneurship... in space!
You are dumb at the internet. You don't know what will go viral. We don't either. But we are slighter less dumber. So here's a bunch of stuff we learned that will help you be less dumb too.
To help the curious class stay relevant, we’ve assembled an A-Z glossary of what we predict to be the 100 must-know terms and concepts for 2017.
We hope this cultural crib sheet will help prepare you for the year ahead.
Enjoy!
Digital Strategy 101 is an overview of the current state of digital strategy and an exploration of core concepts, deliverables, and thought-leaders relevant to young practitioners.
The What If Technique presented by Motivate DesignMotivate Design
Why "What If"...?
The What If Technique tackles the challenge of engaging a creative, disruptive mindset when it comes to design thinking and crafting innovative user experiences.
Thinking disruptively is a disruptive thing to do, which means it's a very hard thing to do, especially when you add in risk-averse business leaders and company cultures, who hold on tight to psychological blocks, corporate lore, and excuse personas that stifle creativity and possibilities (see www.motivatedesign.com/what-if for more details).
The What If Technique offers key steps, tools and examples to help you achieve incremental changes that promote disruptive thinking, overcome barriers to creativity, and lead to big, innovative differences for business leaders, companies, and ultimately user experiences and products.
Let's find out what's what together! Explore your "What Ifs" with us. See www.motivatedesign.com/what-if for details about the What If Technique, studio workshops, the book, case studies and more downloads--including a the sample chapter "Corporate Lore and Blocks to Creativity"
Connect with us @Motivate_Design
An impactful approach to the Seven Deadly Sins you and your Brand should avoid on Social Media! From a humoristic approach to a modern-life analogy for Social Media and including everything in between, this deck is a compelling resource that will provide you with more than a few take-aways for your Brand!
SEO has changed a lot over the last two decades. We all know about Google Panda & Penguin, but did you know there was a time when search engine results were returned by humans? Crazy right? We take a trip down memory lane to chart some of the biggest events in SEO that have helped shape the industry today.
Inside this guide, you'll learn an insiders tips and techniques to getting into the marketing industry - no job applications necessary.
You'll learn what marketing really is, why you'll find a job easily, what entry level marketing jobs look like and four actionable things you can try right now to help get you into the marketing industry.
Visit Inbound.org and the Inbound.org/jobs community jobs board to find opportunities and connect with professional marketers from all over.
The following presentation contains helpful information regarding Radioimmunoassay (RIA) and Enzyme-Linked Immunosorbent Assay (ELISA), including their history, introduction, advantages, procedures and applications.
A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.
Principle, Advantages and Disadvantages Of RIA , Requirements for RIA , Preparation , Development of the Assay system, Assay Procedure , ELISA , Advantages and Disadvantages of ELISA , Types of ELISA, Applications of Immunoassays
Detailed idea on nanotechnology, nanomedicine, types, uses, pharmacotherapy, and future prospects of the nanotechnology. Drug delivery systems, Pharmacokinetics and pharmacodynamics of the nanoparticles are dealt in detail
An intensive material on the anticancer agents. Detailed idea of the various classes of anticancer and recent advances in each class. Newer anticancer drug delivery systems and the anticancer vaccines are also dealt in detail.
An intensive material on recent advances on contraception including the current contraceptive methods and a brief overview on immunocontraception and contraceptive vaccines
A concise overview of pharmacoeconomics, health economics, various costs, various pharmacoeconomic study designs and its application in the field of medicine and drug development
A concise overview of biased agonism, mechanism, beta arrestin pathway, types, examples, GPCR, pros and cons of biased agonism, beta blockers and angiostensin receptor in biased agonism.
Receptor types, mechanism, receptor pharmacology, drug receptor interactions, theories of receptor pharmacology, spare receptors and new concepts like biased agonism
Regulation in clinical trial, Schedule Y and recent amendmentsDr. Siddhartha Dutta
Regulatory framework of India, Acts and Regulations for conduct of clinical trial in India, Schedule Y, approval of new chemical entity and recent amendments
CDSCO and Phamacovigilance {Regulatory body in India}NEHA GUPTA
The Central Drugs Standard Control Organization (CDSCO) is India's national regulatory body for pharmaceuticals and medical devices. Operating under the Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, the CDSCO is responsible for approving new drugs, conducting clinical trials, setting standards for drugs, controlling the quality of imported drugs, and coordinating the activities of State Drug Control Organizations by providing expert advice.
Pharmacovigilance, on the other hand, is the science and activities related to the detection, assessment, understanding, and prevention of adverse effects or any other drug-related problems. The primary aim of pharmacovigilance is to ensure the safety and efficacy of medicines, thereby protecting public health.
In India, pharmacovigilance activities are monitored by the Pharmacovigilance Programme of India (PvPI), which works closely with CDSCO to collect, analyze, and act upon data regarding adverse drug reactions (ADRs). Together, they play a critical role in ensuring that the benefits of drugs outweigh their risks, maintaining high standards of patient safety, and promoting the rational use of medicines.
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NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
The Gram stain is a fundamental technique in microbiology used to classify bacteria based on their cell wall structure. It provides a quick and simple method to distinguish between Gram-positive and Gram-negative bacteria, which have different susceptibilities to antibiotics
Adv. biopharm. APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMSAkankshaAshtankar
MIP 201T & MPH 202T
ADVANCED BIOPHARMACEUTICS & PHARMACOKINETICS : UNIT 5
APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMS By - AKANKSHA ASHTANKAR
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
2. Outline
• Introduction
• Types of assays
• Characteristics of a good assay
• Chemical assays and techniques
• Immunological assays and techniques
• Microbiological assays and techniques
• Bioassay
• Conclusion
3. Assay
• An assay is an investigative procedure 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
4. Types of Assays
• Chemical assays
• Immunoassays
• Microbiological assays
• Bioassay
5. Characteristics of a good
assay method
• Sensitivity
• Specificity
• Repeatability
• Reproducibility
• Validity
• Stability – tissue has to stay “bioassay-fit
5
6. Chemical assays
• A chemical assay refers to the
analysis of a sample material,
called analyte, using a set of chemical procedures
• Qualitative- extraction, distillation, precipitation and other
methods that determine physicochemical properties
• Quantitative- volume or weight of the substance
8. Photometry
• When light is passed through a coloured solution, certain
wavelengths are selectively absorbed giving a plot of the
absorption spectrum of the compound in solution
• The light that is not absorbed is transmitted through the
solution and gives the solution its colour
• Transmittance (T)
• Absorbance (A)= log1/T
• Beer-Lambert law
9. Colorimetry
• Colourless compounds are converted into coloured compounds
using chemical reactions under defined reaction conditions
• The quantity of colour formed is proportional to the quantity of
the original colourless compound
• Colorimeter
• Pros- inexpensive, quantitative estimation of colored
compounds, easily transportable
• Cons- colorless compounds, UV/IR regions, specific
wavelength
• Uses- protein, glucose estimation in various biochemical
samples
14. Flame photometry
• Principle- Matter absorbs light at same wavelength at which it
emits light
Adv-simple/inexpensive/specific/sensitive
to even ppm
Dis-conc cant be measured/high conc-
wrong result
15. Chromatography
• Differential affinities of the various components of the analyte
towards the stationary and mobile phase results in the
differential separation of the components.
Mobile phase or carrier solvent moving through the column
Stationary phase or
adsorbent
substance that stays fixed inside the
column
Eluent fluid entering the column
Eluate
fluid exiting the column (that is collected
in flasks)
Elution
the process of washing out a compound
through a column using a suitable solvent
Analyte
mixture whose individual components
have to be separated and analyzed
16. Column chromatography
• Adsorption chromatography
• Partition chromatography
• Adv- wide variety of mixture can be separated
• Disad- time, plenty of mobile phase required, expensive
17. Paper chromatography
• Ascending
• Descending
• Rf= dis travelled by compound/dis travelled by solvent
• Adv- simple/rapid/inexpensive
• Disadv- small amount can be tested
19. Gas chromatography
• Mobile phase is a gas such as helium and the stationary phase
is a high boiling point liquid adsorbed onto a solid
• Time taken for a particular compound to travel through the
column to the detector is known as its retention time
20. High performance liquid chromatography
• Normal phase
• Reverse phase
• Adv- sensitive to very less conc.(ppt), short time, high
resolution better separation, highly reproducible results
22. An immunoassay is a test that uses antibody and antigen
complexes as a means of generating measurable result
or
An immunoassay is an analytical method which uses
antibodies as reagents to quantitate or detect specific
analytes
26. Competitive Format
• In competitive formats, unlabelled analyte (usually antigen)
in the test sample is measured by its ability to compete
with labeled antigen in the immunoassay.
• The unlabeled antigen blocks the ability of the labeled
antigen to bind because that binding site on the antibody is
already occupied.
27. One step competitive format
• Both the labeled antigen reagent (Ag*) and the unlabeled specimen
(or test sample analyte) compete for a limited amount of antibody.
28. Two step competitive format
• The antibody concentration of the reaction solution is present in
excess in comparison to the concentration of antigen
• Antibody reagent is first incubated with specimen containing
antigens of interest; then in the second step, labeled antigen is
added
• Improved assay sensitivity compared to one step assay formats
29.
30.
31. Noncompetitive (Sandwich) Method
• “Sandwich” assay because analyte is bound (sandwiched)
between two highly specific antibody reagents
• Provides highest level of assay sensitivity and specificity
• Applied to the measurement of critical analytes such as cardiac
and hepatitis markers
32.
33. • One step or two step methods
• The two step assay format employs wash steps in which the sandwich
binding complex is isolated and washed to remove excess unbound
labeled reagent and any other interfering substances
34. Homogeneous and Heterogeneous
Immunoassay Methods
• Immunoassay methods that require separation of bound Ab-Ag*
complex are referred to as heterogeneous immunoassays. Those that
do not require separation are referred to as homogeneous
immunoassays.
• Homogeneous methods have been generally applied to the
measurement of small analytes such as abused and therapeutic drugs.
37. ELISA (Enzyme-Linked Immunosorbent
assay)
• Enzyme immunoassay
• Both qualitative and quantitative measurement of Ag-Ab
binding
• Direct, Competitive, sandwich ELISA- Ag measurements
• Abs- indirect ELISA
• Advantages (sensitivity, ease of handling multiple samples)
without the disadvantages of dealing with radioactivity (like in
RIA)
38. Prerequisites
• Purified antigen (to detect or quantify antibody).
• Purified antibody (detect or quantify antigen).
• Standard solutions (positive and negative controls).
• Sample to be tested.
• Microtiter dishes: plastic trays with small wells in which the assay is
done.
• Wash fluid (buffer).
• Enzyme-labeled antibody and enzyme substrate.
• ELISA reader (spectrophotometer) for quantitative measurements.
40. Performing the Test
• The tubes are filled with the antigen solution (e.g., urine) to be
assayed. Any antigen molecules present bind to the
immobilized antibody molecules.
• The antibody-enzyme conjugate is added to the reaction
mixture. The antibody part of the conjugate binds to any
antigen molecules that were bound previously, creating an
antibody-antigen-antibody "sandwich".
• After washing away any unbound conjugate, the substrate
solution is added.
• After a set interval, the reaction is stopped (e.g., by adding 1 N
NaOH) and the concentration of colored product formed is
measured in a spectrophotometer. The intensity of color is
proportional to the concentration of bound antigen.
41.
42. Isotopic immunoassay
• Based on competition for antibody between radioactive
indicator antigen and unlabelled antigen in test sample.
• Increase in count of unlabeled antigen in test sample decrease
the labeled antigen in bound.
• The concentration of the test antigen can be determined by
comparison with a standard calibrated curve with known
concentration of purified antigen.
43. Nonisotopic
immunoassay
Differ from isotopic immunoassay in:-
o Type of label used
o Means of end point detection
o Possibility of eliminating a separation test
Two types of nonisotopic immunoassay
are:-
o Fluoroimmunoassay
o ELISA (Enzyme-Linked Immunosorbent assay)
44. Radioimmunoassay
• Principle- competitive binding of radiolabelled antigen &
unlabelled antigen to a high affinity antibody
• Involves the separation of a protein (from a mixture) using the
specificity of antibody - antigen binding and quantitation using
radioactivity
• Adv- faster, higher sensitivity/specificity
• Disadv- health hazard, short shelf life, expensive instrument &
needs purified antigen and antibody
45. The technique
• A mixture is prepared of
o radioactive antigen
• Because of the ease with which iodine atoms can be
introduced into tyrosine residues in a protein, the
radioactive isotopes 125I or 131I are often used.
o antibodies against that antigen.
• Known amounts of unlabeled ("cold") antigen are added to
samples of the mixture. These compete for the binding sites of
the antibodies.
46. • At increasing concentrations of unlabeled antigen, an
increasing amount of radioactive antigen is displaced from
the antibody molecules.
• The antibody-bound antigen is separated from the free
antigen in the supernatant fluid, and the radioactivity of
each is measured
47. • The main drawbacks to radioimmunoassay are the expense
and hazards if preparing and handling the radioactive
antigen.
• Both 125I or 131I emit gamma radiation that requires special
counting equipment;
• The body concentrates iodine atoms — radioactive or not —
in the thyroid gland where they are incorporated in thyroxine
(T4).
48.
49. • After determining the ratio
of bound to free antigen
in each unknown, the
antigen concentrations
can be read directly from
the standard curve.
50. Fluoroimmunoassay
• Fluorescent compound in labeling the antibodies and antigen
• Europium fluoresces when it excite by light in the presence of a
developing solution
• It is 10 times more sensitive than RIA
51. • A modern fluorescent based immunoassay uses as the
detection reagent a fluorescent compound which
absorbs light or energy (excitation energy) at a specific
wavelength and then emits light or energy at a different
wavelength
• The difference between the wavelength of the excitation
light and the emission light is called the Stokes shift.
52. Application of immunoassay
in food Industry
• Many of the macromolecule that can found in food are
good antigen and antibodies are capable of recognizing
them and small molecule.
• Composition of raw material and final product
• Harmful and useful minor substances with biological
activity (toxins and allergens)
• Enzyme detection
• Contaminants detection and determination (hormones
,drug, pesticides residue)
53. Microbiological assays
• Principle- Based upon a comparison of the inhibition of
growth of micro-organisms by measured concentration of the
antibiotics to be examined with that produced by known
concentrations of a standard preparation of the antibiotic having
a known activity
1. The cylinder-plate
(or cup-plate) method
2. The turbidimetric
(or tube assay) method
57. Definition
Comparative assessment of relative potency of a test compound to a
standard compound on a living or biological tissue
Quantitative measurement of the amount of active principle or
substance in a pharmaceutical preparation or biological material using
a suitable biological system
58. Comparison Of Chemical
& Bioassay
Bioassay
Less Precise
More time consuming
Active constituent &
structure not known.
More sensitive
Chemical Assay
More Precise
Less time consuming
Active constituent &
structure fully established.
Less sensitive
59. Indications Of Bioassay
• Chemical method is either
Not available
If available, too complex,
Insensitive to low doses e.g. Histamine
• If active principle of drug is not known e.g. insulin
• To measure the pharmacological activity of new or
chemically undefined substances
• Chemicals with similar structure, but different biological
activity
60. • Chemical structure known; cannot be actively purified. Eg:
Peptide hormone
• Active principle cannot be isolated e.g. posterior pituitary
extract, insulin
• To compare the strength of a drug obtained from various
sources due to different compositions (Eg:Cardiac
glycosides,catecholamines)
• Biological activity of drug cannot defined by a chemical assay
e.g. Cis and Trans form of methyl phenidate.
• For biological standardization of drugs obtained from natural
sources as these cannot be obtained in pure form. Eg:
Oxytocin, Vasopressin, Insulin, Heparin..
61. Principles of bioassay
• To compare the test substance with the International Standard
preparation of the same
• To find out how much test substance is required to produce
the same biological effect, as produced by the standard
• Activity assayed should be the activity of interest
• Standard & test sample - similar pharmacological effects &
mode of action
62
62. • both should be compared for their established
pharmacological effect using specified technique
• Ex: *Ach – contractile response on frog rectus
• *Histamine – contractile response on guinea pig ileum
• Problem of biological variation must be minimized
Experimental conditions - kept constant
Animals - same species, sex and weight
Number of animals - large enough to minimize error (individual
variation)
Isolated preparations - sensitive
63
63. Prerequisites for Bioassay
• Physiological salt solutions
• Kymograph: Sherrington- starling kymograph
• Student Organ bath
• lever
65. 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 précise
• Employed for:
• Comparison of LD50 and ED50
66. Graded Bioassay
Effect is produced gradually depending on dose.
E.g. Contraction of smooth muscle preparation
67. 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.
68. Various Physiological salt solutions
Frog-
Ringer
Kreb’s Tyrode Ringer-
Locke
De
Jalon
Mc
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.
H²O
0.1 g 1.4 g 0.5 g --- --- 1.4 g
NaHCO³ 2 g 21 g 10 g 1.5 g 5 g 21 g
CaCl² 1.2 g 2.8 g 2 g 2.4 g 0.6 g 2.4 g
Glucose 20 g. 20 g. 10 g. 10 g. 5 g. 20 g
Aerating
Gas
air O² +
5%CO²
O² or
air
Pure O² O² +
5% CO²
O² + 5%
CO²
For 10 litres
pH- 7.3-7.4
•Calcium chloride to be added last.
•Calcium chloride and magnesium chloride are hygroscopic, so use stock
69. Uses: Physiological salt Solutions
Physiological salt
solutions
Uses
Frog-Ringer Amphibian tissue preparation
Kreb’s Mammalian/Avian skeletal
muscle preparation
Tyrode Intestine preparation
Ringer-Locke Heart muscle preparation
De Jalon Rat uterus preparation
71. Step 2: Arrange the instrument and
adjust the water bath.
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
72. Student Organ bath
• Outer bath:-
First designed by rudolph
magnus
Perpex glass
Store water outside the inner
bath to maintain the
temperature
• Inner bath:-
o Glass
o To observe the tissue during
experiment
o 5-50ml (usually 10ml)
73. • Tissue holder and oxygen supply:-
Tissue is attached inside the inner water bath to a tissue holder.
Also supports the oxygen supply to the tissue.
74. Step:3 -Balance the lever
• Lever:
Three basic parts:
• Effort arm- where force in applied
• Load arm- where effect of force is
observed
• Fulcrum
Classes of lever – 3
Types of lever
75. • Magnification :
= Distance from the fulcrum to the writing point
Distance form the fulcrum to the tied tissue
o For slow contracting muscles:- 10-15 times
o For fast contracting muscles:-5-10 times
French essai "trial", and the noun assay thus means "trial, test of quality, test of character“
(analytic)
in laboratory medicine, pharmacology,
environmental biology, and molecular biology
Specificity- ability of the test/ assay to detect true negatives
Repeatability: Same observation using same instruments and operators, and over short time periods
Reproducibility: Same observation using different instruments and operators, and over longer time periods
Validity- extent to which a test measure what it is suppose to measure(Most imp criteria)
Stability- same dose same response same set up
Accuracy- diagnostic effectiveness/ ability to differentiate +/-cases correctly
Precision
Branch -analytical chemistry,
objective is to identiFy of the analyte's unknown components,
isolate and quantify them
utilizes instruments and techniques, such as spectroscopy, chromatography and electrophoresis, to measure the physical quantities of the analyte.
Science of measuring light
VY
BO
GR
Biochemical samples for estimation
Hb , identify sunstandard/ counterfeit drug
Test water quality and find out various chemical/ ions
uses the basic principles of photometry but the solutions have to be coloured, i.e. they must absorb light in the visible range.
Ordinary light from a tungsten lamp is passed through a suitable filter to obtain light of a desired wavelength, which is then passed through the solution.
Transmitted light falls on the sensitive surface of selenium photocell which generates a current proportional to the light intensity. The cell is connected to a galvanometer, which is used to read out percentage transmission or absorbance.
Detection of impurities
Kinetics of a chemical reaction
Sturucture of an organic molecule
Presence or absence of functional groups
Molecular wt detection
Analytical methods depending on ultraviolet absorption Examples include serum enzyme assays,
assays of glucose, urea, uric acid,
advantage of the UV absorption of the coenzymes NADH and NADPH at 340nm.
Device used to measure parameters of fluorescence: intensity and wavelength
FLU spectrum- characteri of molecule
Liquid chromatography
Vit b 1 and 12
Analysis of organic and aromatic molecules
Nuclear research- uranium
Inorg-ru, al, cad, boron789
Flame atomic emission spectrometry
Wavelength- what element/quali
Color intensity- conc/quanti
Dis-mol sturucture/carbon/H2/ halide not determined
Affinity(strength of adhesion, in turn, is dictated by two properties of the molecule: ‘Adsorption’ and ‘Solubility’.
Used for separation, purification, identification of compounds in a mixture
Stationary phase= silica, activated alumina, cellulose, ion exchange resins
Can be a solvent- partition chromato
Mobile phase- liquid or gas
Estimation of drugs
Separation and purification of of tanin/glycosides/resin/flavinoid
Used for small quantity of sample
Separation & identification of mix of drugs
Analysis of metabolites in urine, blood
Vitamins, antibiotics, proteins
Small quantity sample
If invisible then spray ninhydrin dye- brown/purple colour to amino acids
Or incorporate fluroscence material in the adsorbent and see with uv light to differentiate the spots
retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. high boiling point means a long retention time. High solubility in the liquid phase means a high retention time high column temperature shortens retention times for everything in the column
High pressure
Column can be used without waiting for regenration
analytes --naturally present in the body (such as a thyroid hormone)
produceD but are not typically present (such as a cancer antigen), or
do not naturally occur in the body (such as an abused drug).
“Immuno” refers to an immune response that causes the body to generate antibodies.
“Assay” refers to a test.
An immunoassay is a test that uses immunocomplexing when antibodies and antigens are brought together.
antibody is a protein that is produced by the body in response to an “invading” (foreign) substance.
An analyte is anything measured by a laboratory test. antibody, or an antigen.
label is a molecule that will react as part of the assay, so a change in signal can be measured in the blood:reagent solution
a radioactive compound,
an enzyme that causes a change of color in a solution,
or a substance that produces light.
Antibodies are basically immunoglobins that bind to different natural and synthetic antigens in the body such as carbohydrates, lipids, proteins and nucleic acid
especially suited for the analysis of compounds at low concentration and in
samples with little or no preparation, since their
detection limits are usually within nanogram to picogram range.
Remember that in the competitive format, less bound labeled antigen indicates more antigen present in the test sample.
assay formats provide several fold.
in a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present.
The amount of antigen in the test sample is inversely related to the amount of label measured in the competitive format.
The two step noncompetitive format usually offers the highest specificity and sensitivity of all the assay formats discussed here.
Since homogeneous methods do not require the separation of the bound Ab-Ag* from the free Ag*, they are generally much easier and faster to perform.
first described by Engvall and Perlmann in 1971detect antigen (hormones, enzymes, microbial antigens, illicit drugs) or
antibody (anti-HIV ) in body fluids or tissue culture supernatents
requires: Ab fixed to a solid surface(inner surface of a test tube)
a prep of same ab coupled to an enzyme. This is one (e.g., β-galactosidase) that produces a colored product from a colorless substrate.
Sandwich method
Adv- 2-5x sensitive, no purification of sample
Disadv- full knowledge of antigen needed and finding 2 epitopes needs expertise
As antigen is found by entrapment b/w 2 ab so its very specific
detecting allergens in food and house dust
measuring toxins in contaminated food
The amount of radioactivity measured is indicative of the amount of analyte present.
Fluorescein is a fluorescence label that absorbs light at 490 nm and releases this energy at 520 nm.
Radioisotopes and enzymes were replaced by new
Emission duration is short but less than that of the background noise. can be measured by special instrument which is unfortunately very expensive.
B12- organism is inoculated into a medium containing all the growth factors needed except the one under examination;
the rate of growth is then proportional to the amount of this nutrient added in the test substance.
American Type Culture Collection (ATCC).
Dipotassium Hydrogen Phosphate,
K2HPO4
Potassium Dihydrogen phosphate, KH2PO4
Active principle of drug is unknown
Active principle 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 assayed in microgram conc.
Chemical composition of drug is different but has same pharmacological action e.g. cardiac glycosides isolated from diff sources, catecholamines etc.
To measure LD 50 and ED 50
For biological standardization of drugs from natural sources which cannot be obtained in a chemically pure form e.g., vasopressin, oxytocin, insulin, heparin
Not possible to separate interfering substance e.g. Vitamin D.
For biological standardization of drugs from natural sources which cannot be obtained in a chemically pure form e.g., vasopressin, oxytocin, insulin, heparin
The standards are internationally accepted samples of drugs maintained and recommended by the Expert Committee of the Biological Standardization of W.H.O.
In India, standard drugs are maintained in Government institutions like Central Drug Research Institute, Lucknow, Central Drug Laboratory, Calcutta, etc
All bioassays should be comparative against a standard drug
Standard & new drug should be as far as possible identical to each other
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.
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, 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.)
Elicits an ‘All or None’ response in different animals
E.g.
Digitalis induced cardiac arrest in guinea pigs
Hypoglycaemic convulsions in mice.
Digitalis induced head drop in rabbits
Graded responses to varying doses
Unknown dose response measured on same tissue