Radioimmunoassay (RIA) is a sensitive technique that uses radioactively labeled molecules and antibodies to detect trace amounts of substances. It works by measuring the competition between a radioactive antigen and a non-radioactive antigen for binding to a limited number of antibodies. RIA has applications in measuring hormones, vitamins, drugs, and markers of infection or cancer. It provides high specificity and sensitivity, allowing detection of picogram quantities. However, it requires special handling of radioactive materials and trained personnel.
This document describes detailed information about Radio immuno assay (RIA) including its principle, procedure, advantages, disadvantages, application etc
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
This document describes detailed information about Radio immuno assay (RIA) including its principle, procedure, advantages, disadvantages, application etc
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
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
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation.
The support medium is a filter paper
The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution, through which electric current is passed.
Frequently used in isolating proteins, amino acids and oligopeptides.
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
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation.
The support medium is a filter paper
The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution, through which electric current is passed.
Frequently used in isolating proteins, amino acids and oligopeptides.
In this slide discuss about Radioimmunoassay and it will help to understand about assay details.
If any query feel free to contact to me.
If any suggestion please share with me.
This presentation explains about the Immunoassay ,radio immuno assay, definition, types, Principle , procedure, steps involved ,advantages ,disadvantages ,Application, RIA in insulin. RIA in Digitalis drug ligand etc....
SYNOPSIS
INTRODUCTION
PRINCIPLE
HISTORY
HOW TO RIA WORK
METHOD
APPLICATION OF RIA
ADVANTAGE
DISADVANTAGE
CONCLUSION
REFERENCES
The technique in which a radioisotope is used as a tag or label (i.e. radioisotope covalently linked to antigen or antibody) for the detection of antigen-antibody complex is known as RIA.
RIA involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantifitation using radioactivity.
RIAs utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. 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.
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Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
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Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
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Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
1. Prepared By
Dr.J.Swaminathan M.Pharm.,MBA., Ph.D.
Associate Professor,
ADHIPARASAKTHI COLLEGE OF PHARMACY,
MELMARUVATHUR – 603 319
RIA
( Radio Immuno Assay)
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 1
6. Radioimmunoassay (RIA) is a scientific method used to test antigens (for
example, hormone levels in the blood) without the need to use a bioassays.
Radioimmunoassay (RIA) is a Radio-analytical technique with remarkable
sensitivity and a high degree of specificity that is widely used for the estimation
of a variety of molecules present in complex matrices. Also known as Radio
tracer technique and best example of invitro diagnosis technique using radio
isotopes.
This technique is used over a wide spectra of substances such as hormones,
steroids, vitamins, drugs, tumor markers and viral antigens.
Radio Immuno Assay
Use of radio
active material
Antigen antibody
binding theory
Detection of
compoundRADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 6
8. This isotopic measuring method was developed in 1959 by two Americans,
biophysicist Rosalyn Yalow and physician Solomon A. Berson.
RIA combines the specificity of an antigen-antibody reaction with sensitivity of
radioactivity measurements.
This is a technique used for detection of micro quantities of protein, viral antigens,
antibodies, structural proteins, vitamins and drug and their metabolites.
It can also be used for detection of pictogram quantities (10−12 g) of biological
constituents present in biological fluid.
RIA is used in place of bioassay in various branches of science like Biochemistry,
Microbiology, and Hematology and Clinical pharmacology.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 8
10. RIA works on basic principle of biochemistry that competitive binding between
antigens for same antibody binding site.
The competition of an analyte with its radioisotopically labeled counterpart for a
limited amount of antibody, the specific reagent, is the underlying principle of this
technique. Increasing the analyte concentration inhibits the binding of the labeled
analyte to the antibody.
Ag + Ag* + Ab AgAb + Ag*Ab + Ag
Unbound Ag* and Ag washed out
Radioactivity of bound residue measured
Ligand conc. is inversely related to radioactivity
Ag : ligand to be measured ;
Ag*: radio labelled ligand
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 10
11. A
B
C
Antibody
Unlabeled antigen
Labeled antigen
Now how the competition occur with increase the concentration of unlabeled
antigen in the system of RIA in three different cases A, B and C.
Here first antibodies bound with labeled antigen are put into the known
concentration of analyte solution and it is observed how the labeled antigen free
from antibody and unlabeled will bind in place of there.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 11
12. The concentration of the unknown analyte is thus obtained by comparing its
inhibitory Effect on the binding of the labeled analyte to that of a known standard.
After determining the ratio of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve (as shown above
After determining the ratio of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve (as shown above).
1
2
1. – Ratio in unknown and
2. - Antigen in unknown
Red line – binding line
Green line – free labeled antigen
RADIO IMMUNO ASSAY - DR.J.SAMINATHAN, APCP, MLMR12
13. From graph we can also calculate %F (fraction of free labeled antigen) and %B
(fraction of bound labeled antigen).
&
• F – amount of free labeled antigen
• B - amount of bound labeled antigen
Here as the concentration of unlabeled increase it replaces the labeled bound
antigen by competitive binding it inhibiting the binding of labeled one.
Antigen- antibody complex depend on antibody affinity (Ka) –
The affinity with which antibody binds antigen results from a balance between
the attractive and repulsive forcesRADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 13
14. Advantages
Highly specific: Immune reactions are specific,the greater the specificity of the
antiserum, the greater the specificity of the assay.
High sensitivity : Immune reactions are sensitive, Using antibodies of high
affinity it is possible to detect a few picograms (10−12 g) of antigen in the tube.
Accuracy and Precision
Disadvantages
Radiation hazards: Uses radio labelled reagents
Requires specially trained persons
Labs require special license to handle radioactive material
Requires special arrangements for
Requisition, storage of radioactive material
radioactive waste disposal.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 14
15. Development of the
Assay System
Development of the Assay System
1. A crucial step is separation of unbound antigens
2. This achieved by binding the antibodies to the microtitre well surface
[Solid phase RIA]
3. Antigens bound to the fixed antibodies remain stuck to the inner surface
4. Decanting & washing the well removes unbound antigens
5. Other techniques of separation: Centrifugation
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 15
16. Assay Procedure
1. Add known amounts of the test sample + labelled
antigen into the microtitre wells
2. Incubate allow the reaction to reach completion
3. Decant & wash contents of the well removes all
unbound antigens
4. Radioactivity remaining in the Microtitre wells
measured by a Counter [GM counter , Scintillation
counter etc]
5. Intensity of radioactivity is inversely correlated with
the conc of antigens in the test sample
6. Sensitive to very low conc of antigens
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 16
20. 1. Radio labelling of the Antigen or radio labelled production
2. Preparation & characterisation of the Antigen [Ligand to be
analysed]
3. Preparation of the Specific Antibody
4. Development of Assay System or separation techniques
Methods in RIA :
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 20
21. 1. Preparation and radio labeling of antigen
Antigen preparation:
Synthesis of the molecule
Isolation from natural sources
Radiolabelling [Tagging procedure] :
Two most commonly used radio labels in RIA 3 H and 125 I although Se, P, Co
14 C and 131 I have also been used. but these have some limitations that are –
32P and 57Co = limited by stereo chemical aspects because drug
naturally contain phosphorous and cobalt
14 C = low specific activity field
131 I = Short decay half life and radio degradation character
on the other hand 3 H and 125 I have advantages as follows:
3 H = Direct incorporate into molecular structure, half lives(12 years)
125 I= High activity and ease of counting (half lives 60 days)
75 32 57
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 21
22. Technique of labeling of drug or antigen with 3H and 125I:
Specific tritium labels are generally obtained by reducing an appropriate
precursor in presence of 3H.
Labeling of drug with 125I include chloramines-T, monochloride exchange
and enzymatic iodination methods. The most suitable iodination
procedure is depending on the stability of the drug and the specific activity
that is sufficient to meet the sensitivity requirements of assy.
Comparison of 3H and 125I is as follow:
Sr no. Tritium (3H) Iodine (125I)
1 It is more efficient when relatively small
numbers of samples are assayed.
It is more efficient as the numbers of samples are
increased.
2
Such type of problem does not occur.
It’s having quality control problems such as
damage during the reaction and radiation damage
following synthesis.
3 Having advantages of long half life, higher
affinity.
It has short half life dictates frequent preparation.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 22
23. it is possible to find the specific antibody for drug(antigen) as a result of
sensitivity reaction
Several drugs which have induced antibodies production due their inherent
antigenicity like – penicillin, strychnine, tetracycline, sulphonamide and
procainamide. However their low specificity and limited availability makes their
use rather improbable.
In most cases the drugs (analysed antigen) are bind with suitable carrier protein
to make conjugated antigen immunogenic.
There are several reactive groups on protein carrier which can used for the
purpose of conjugation of standard drug(antigen).these groups include the
terminal amino and carboxyl groups, ε-amino group of lysine, the carboxyl group
of aspartic and glutamic acid, the phenolic group of tyrosine.
2. Preparation purification of drug-protein conjugate(ligand-antigen) to be
analyzed:
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 23
24. The most commonly used methods for conjugation are as follows:
Carbodimide and glutaraldehyde reaction
Carbony-diimidazole reaction
Scotten-baumann reaction
Mannich reaction
Diazotization reaction
The method chosen for conjugated will depend on the functional groups
available for coupling the drug to protein and no. of drugs molecules
which are to be coupled to protein.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 24
25. Preparation :
Once the pure antigen prepared then it is emulsified into equal volumes of
saline and Freud’s adjuvant (contain alum ppts, natural detergents, mineral oils,
killed mycobacterium) to get final concentration of 10 to 50mg/ml.
One ml of the emulsion is injected intradermally, subcutaneously, and/or
intramuscularly at weekly or monthly intervals into multiple sites of a suitable
animal species such as rat, guinea pigs or rabbits. A suitable animal species is
usually dictated by the size of the animal facilities.
Animals are tested after 3-4 weeks and then blood is collected and separated.
The resulting blood containing antibody called antiserum. It is directly used in
assay and should be stored at 4°c.
Characterization:
Characterization of antiserum done by fractionation, immunoadsorption or
immunosaturation technique.
3. Preparation & characterization of the Specific and high affinity Antibodies:
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 25
27. 4. Development of suitable separation techniques to separate free from
bound standard drug:
Several methods that employ physiochemical and immunological separation have been
devised as follows –
Physical methods: Filtration, Chromatography, Electrophoresis, charcoal- dextran
adsorption, and ion exchange resin.
Disadvantages: it is tend to be time dependent and harsh so they may remove bound drug
from antibody during separation.
Chemical method: organic solvents such as ethanol, dioxane and polyethylene glycol
(PEG), and salts such as sodium, zinc, ammonium sulfate.
Disadvantages: chemical precipitation may precipitate free as well as bound drug during
separation depending on physicochemical nature of drug.
Second antibody method: it is most physiologic procedure to precipitate bound antigen.
This method employs, an antibody against gamma globulin of the animal species used to
produce antidrug antibody to the plasma serum to precipitate drug-antibody complex.
Disadvantages: This technique have required prolong incubation time.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 27
29. Other related Immuno Assay:
Related methods include the use of antibodies to specifically bind antigenic
substances.
They do not include competitive protein binding assays (CPBA) or receptor site
binding assay (RSBA).
The most commonly related method is Immunoradiometric (IRM) – the
technique which employs an excess of labeled antibody to quantify the drug in the
system. The principle of the system displayed in figure as below.
S + Ab٭ SAb٭ + Ab٭ S Ab٭ + SPAb٭
SP
S is unknown or standard antigen, Ab٭ is labeled antibody, and SP is the antigen
coupled to a solid support.
SAb٭ fraction will remain in the supernatant, where as the SPAb٭ will be coupled
to the solid support.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 29
30. •Advantages:
The antibody can be more readily labeled with high specific activity using 125I.
No need of any specific separation technique as in RIA.
Instead of using radio tracers several other methods rely on alternate labeling
procedures. They include hemaglutination-inhibition, fluorescence, and free radical
or spin label and last Enzyme Immuno-Assays.
Only Enzyme Immuno-Assays (EIA) – has gained wide acceptance. The
theoretical concept of EIA is same as RIA, principle of EIA is as follows:
P S+٭ Ab S٭Ab
+
S SAb
S and S٭ are pure and enzyme linked
drug, respectively; Ab is antibody; and P is
the measurable reaction product that can
result only when free S٭ react with an
appropriate antibody.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 30
31. •Advantages of EIA over RIA:
•No radiation hazard
•Economy of quantitation
•Homogenous EIA does not require separation of free and bound ligands
(antigen)
•Disadvantages:
•Relatively insensitive due to the low catalytic activity of enzymes
•A linkage specificity problem when same linkage used for antibody conjugates
and enzyme conjugates.
Several drug EIA systems are currently in use in the clinical chemistry
laboratory, primarily for the detection of drugs of abuse and quantification of
Anticonvulsant and digoxin plasma concentration.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 31
32. Application of RIA:
Analysis of hormones, vitamins, metabolites, diagnostic markers:
E.g. ACTH, FSH, triiodothyronine (T3) and thyroxine (T4), Glucagons, Insulin,
Testosterone, vitamin B12, prostaglandins, glucocorticoids,
Therapeutic drug monitoring:
Barbiturates, morphine, digoxin,
Diagnostic procedures for detecting infection :
HIV, Hepatitis A, B etc
Tumour markers:
• RIA of tumour markers such as alpha-fetoprotein (AFP), carcinoembrionic
antigen (CEA), b-HCG for choroid -carcinoma, prostate specific antigen (PSA) for
prostate cancer, are available for detection and management of cancer
Non clinical application: such as veterinary science, food processing industry,
drug industry, forensic science and environmental monitoring.
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 32