In December of 1898, Marie and Pierre Curie announced the discovery of a second element found in the uranium-extracted residues of pitchblende ore and, due to the intense radiation rays it emitted, it was named radiumThe discovery of radium brought radioactivity to the attention of the general public and inspired many new uses of radioactivity. Radiopharmaceuticals, or medicinal radiocompounds, are a group of pharmaceutical drugs containing radioactive isotopes. Radiopharmaceuticals can be used as diagnostic and therapeutic agents. Radiopharmaceuticals emit radiation themselves, which is different from contrast media which absorb or alter external electromagnetism or ultrasound. Radiopharmacology is the branch of pharmacology that specializes in these agents.

1. Radiochemistry and
Radiopharmaceuticals
Prepared by
ROHAN JAGDALE
YTIP,Mumbai
Guided by
Prof. Dr. VIJETA DHABRE
Pharmaceutical Analysis II
University of Mumbai

2. Contents
❏ Basic terms
❏ Relative biological effectiveness
❏ Radionuclidic purity
❏ Radiochemical purity
❏ Safety aspects of radiopharmaceutical laboratory
❏ Measurement of Radioactivity
❏ Requirements of radiopharmaceutical
❏ Radionuclidic generator
❏ Quality control of radiopharmaceuticals
❏ Radiochemical methods in analysis
❏ Radioimmunossay

3. Introduction
In December of 1898, Marie and Pierre Curie announced the discovery of a second
element found in the uranium-extracted residues of pitchblende ore and, due to the
intense radiation rays it emitted, it was named radium
The discovery of radium brought radioactivity to the attention of the general public
and inspired many new uses of radioactivity.
Radiopharmaceuticals, or medicinal radiocompounds, are a group of pharmaceutical
drugs containing radioactive isotopes. Radiopharmaceuticals can be used as
diagnostic and therapeutic agents. Radiopharmaceuticals emit radiation themselves,
which is different from contrast media which absorb or alter external
electromagnetism or ultrasound. Radiopharmacology is the branch of pharmacology
that specializes in these agents.

4. Radioisotope
Radioisotopes are radioactive isotopes of an element. They can also be
defined as atoms that contain an unstable combination of neutrons and
protons, or excess energy in their nucleus.
An unstable form of a chemical element that releases radiation as it breaks
down and becomes more stable.
Radioactive isotopes have many useful applications. In medicine, for
example, cobalt-60 is extensively employed as a radiation source to arrest
the development of cancer. Other radioactive isotopes are used as tracers
for diagnostic purposes as well as in research on metabolic processes.

5. Radioactive decay
Radioactive decay (also known as nuclear decay,
radioactivity, radioactive disintegration or nuclear
disintegration) is the process by which an unstable
atomic nucleus loses energy by radiation. A material
containing unstable nuclei is considered radioactive.

7. Half - life of radioactivity
Half-life, in radioactivity, the interval of time required for one-half
of the atomic nuclei of a radioactive sample to decay (change
spontaneously into other nuclear species by emitting particles
and energy), or, equivalently, the time interval required for the
number of disintegrations per second of a radioactive

8. Specific activity
Specific activity (a) is the activity of a given radionuclide per unit mass.
Specific activity is the activity per quantity of a radionuclide and is a physical
property of that radionuclide.
i.e radioactivity per unit volume, can be utilized in some radiation
measurements, e.g blood volume determination. In blood volume
determinations, the administered IV dose in CPM/ml is related to the
radioactivity after dilution by the patients.
Specific concentration

9. Becquerel (Bq)
The becquerel is the SI derived unit of radioactivity. One becquerel
is defined as the activity of a quantity of radioactive material in
which one nucleus decays per second.
1 Becquerel is equal to 1 disintegration per second
1 Bq = 1 dps

10. Curie
The curie is a unit of ionizing radiation (radioactivity),
symbolized Ci and equal to 3.7 x 10 10 disintegrations or
nuclear transformations per second. This is approximately the
amount of radioactivity emitted by one gram (1 g) of
radium-226.
The unit is named after Pierre Curie, a French physicist.

11. Sievert
The IS unit for the amount of ionizing radiation required to produce
the same biological effect as one rad of high-penetration x-rays,
equivalent to a gray for x-rays. It measures the radioactive dose
equivalent. One sievert is equal to one gray multiplied by a relative
biological effective factor, Q, and a factor that takes into account
the distribution of the radiation energy, N. The sievert is the correct
unit to use when you wish to monitor the biological danger of
radiation.

12. Gray
The IS unit for the energy absorbed from ionizing radiation, equal to one
joule per kilogram. An absorbed dose of one gray is equal to the
absorption of one joule of radiation energy by one kilogram of matter.The
gray is the correct unit to use when you wish to monitor energy absorbed
per unit mass.
The gray (Gy) is the only actual physical dose ! 1 Gy = 1J/Kg
But the effect of the radiation dose on the body changes with the radiation
type. Beta and Gamma are quite equivalent but Alpha is much more
dangerous.

13. For calculations
● 1 gray (Gy) = 100 rad
● 1 rad = 10 milligray (mGy)
● 1 sievert (Sv) = 1,000 millisieverts (mSv) = 1,000,000 microsieverts (μSv)
● 1 sievert = 100 rem
● 1 becquerel (Bq) = 1 count per second (cps)
● 1 curie = 37,000,000,000 becquerel = 37 Gigabecquerels (GBq)
● For x-rays and gamma rays, 1 rad = 1 rem = 10 mSv
● For neutrons, 1 rad = 5 to 20 rem (depending on energy level) = 50-200 mSv
● For alpha radiation (helium-4 nuclei), 1 rad = 20 rem = 200 mSv

14. Relative Biological Effectiveness
The relative biological effectiveness (RBE) is defined as the ratio of the doses
required by two radiations to cause the same level of effect.Thus, the RBE
depends on the dose and the biological endpoint.
RBE is calculated as a ratio of the reference radiation dose to the dose of the test
radiation
RBE is highly variable and depends on several parameters including the type of
radiation, total dose, dose rate, dose fractionation pattern, and the biological effect
being assayed.

15. Radionuclidic purity
● The fraction of the total radioactivity in the total desired
radionuclide.
● A compound has absolute radionuclidic purity if it contains no
radionuclide other than the one of interest.
● The radionuclidic purity is an essential parameter in the quality
control of radiopharmaceuticals
● Impurities arises from -
● 1. Extraneous nuclear reactions due to isotopic impurities in the
target material.
● 2. Fission of heavy elements in the reactor

16. ● The undesirable radionuclides may belong to the same element
as the desired radionuclide or to a different element
● Impurities can be removed by appropriate chemical methods
● Radionuclidic purity is determined by measuring thee half-lives
and characteristic radiations emitted by individual radionuclides

17. Radiochemical purity
● The fraction of the total radioactivity in the desired chemical form in the
radiopharmaceutical
● Radiochemical purity is important in radiopharmacy since it is the
radiochemical form which determines the biodistribution of the
radiopharmaceutical. Radiochemical impurities will have different patterns
of biodistribution which may obscure the diagnostic image obtained and
render the investgation meaningless.
● The presence of radiochemical impurities in a radiopharmaceutical results
in poor-quality images due to the high background from the surrounding
tissues and the blood, and gives unnecessary radiation dose to the
patient.

18. ▫Radiochemical impurities arises from
1. Decomposition due to action of solvent
2. Change in temperature or pH of light
3. Presence of oxidising or reducing agent
▫Decomposition of labelled compound by radiolysis depends on -
1. The specific activity of radioactive material.
2. The type and energy of the emitted radiation.
3. The half- life of radionuclide.

19. Safety aspects of radiopharmaceutical laboratory
Radiation Saety is a term applied to concepts, requirements, technologies and
operations related to protection of people against the harmful effects of ionizing
radiation.
Safe Handling of Isotopes :
1. GRP needs to be strictly followed for operations with unrelated sources to
reduce the chances of getting unwanted and avoidable radiation exposure.
2. It is necessary to mark the area in which the radio work is carried out and it
should be monitored regularly at periodic intervals.

20. 3. Unnecessary movements of persons or materials should be avoided in the
hot laboratory or radiopharmacy.
4. All the radiation workers must wear suitable protective clothing and
radiation monitoring devices, Surgical gloves is necessary.
5. When not in use, the radionuclides must be kept in sealed containers.
6. The areas should be surveyed regularly for both contamination & exposure
hazards.
7. Work areas should be covered with a plastic, glass or stainless.
8. Tray with absorbent paper should be use to catch any spills and to prevent
the spread of contamination.

21. 9. Do not pipette by mouth
10. Wash hands thoroughly.
11. Radioactive materials should never be touched with hand but handled with
forceps or suitable instruments.
12. Do not eat, drink and smoke in areas where unsealed radionuclides are
stored.
13. The radiation survey meter should be used to ensure safety of workers and
public.
14. Survey an wipe test suggested action levels are
For unrestricted areas 0.25 mR/ hr. ( milli Roentgens)
For restricted area 20 mR/hr.

22. Measurement of Radioactivity
- Geiger - Muller Counting
- Liquid Scintillation Counting

23. Geiger muller counting

24. Introduction
● The Geiger counter is an instrument used for measuring ionizing radiation
● It detects ionizing such as alpha particles, Beta particles and gamma rays
using the ionization effect produced in a Geiger- Múller tube
● It is perhaps one of the world's best know radiation detection instruments.
● The original detection principle was discovered in 1908.
● The development of the Geiger - Múller tube in 1928 that Geiger Múller
counter became a practical instrument.
● It has been very popular due to its robust sensing elements and relatively low
cost

25. Operating principle
● A Geiger - Múller counter consists of a Geiger - Múller tube, the
sensing element which detects the radiation, and the processing
electronics - Results in Display.
● Geiger - Múller tube is filled with an inert gas such as helium, neon or
argon at low pressure, to which a high voltage is applied.
● Tube briefly conducts electrical charge when a particle on photon of
incident radiation makes the gas conductive by ionization.
● The ionization is considerably amplified within the tube by the
Townsend Discharge effect to produce an easily measured detection
pulse.

26. Operating principle
● This large pulse from the tube makes the G-M counter cheap to
manufacture, as the subsequent electronics is greatly simplified.
● The electronics also generates the high voltage, typically 400 - 600
volts
Two types
1. Counter per second
2. Absorbed dose

27. Counter per second
● The number of ionizing events displayed either as a count rate, commonly
“count per second “
● The counts readout is normally used when the alpha or beta particles are
being detected.
Absorbed dose
● More complex to achieve display of radiation dose rate, displayed
in a unit such as the sievert.
● Normally used for measuring gamma or x-rays dose rates

30. How it works?
● Radiation (dark blue) is moving about randomly outside the detector tube.
● Some of the radiation enters the window (gray) at the end of the tube
● When radiation (dark blue) crush with gas molecules in the tube (orange), it
causes ionization : some of the gas molecules are turned into positive ions (red)
and electrons (yellow)
● The positive ions are attracted to the outside of the tube (light blue).
● The electrons are attracted to a metal wire (red) running down the inside of the
tube maintained at a high positive voltage.
● Many electrons travel down the wire making a burst of a current in a circuit
connected to it.
● The electrons make a meter needle deflect and, if a loudspeaker is connected,
you can hear a loud click every time particles are detected.

31. Liquid Scintillation Counting

32. What is Scintillation Counter?
● A scintillation counter measures ionizing radiation.
● The sensor, called a scintillation, consists of a transparent
crystal, usually phospher that fluoresces when struck by
ionizing radiation.
● A sensitive photomultiplier tube (PMT) measures the light from
the crystal. The PMT is attached to an electronic amplifier and
other electronic equipment to count the signals produced by the
photomultiplier.

33. Scintillation counter apparatus
● When a charged particle strikes the scientillator, a flash of light is
produced.
● Each charged particle produces a flash.
● When a charged particle passes through the phosphor some of the
phospher’s atoms get excited and emit photons.
● The intensity of the light flash depends on the energy of the charged
particles.
● Cesium iodide ( CsI) in crystalline form is used as the scintillator for the
detection of protons and alpha particles ; sodium iodide (Nal) containing a
small amount of thallium is used as a scintillator for the detection of
gamma waves.

34. Liquid scintillation counting
● Samples are dissolved or suspended in a “cocktail “ containing an
aromatic solvent and small amounts of other additives known as
fluors.
● Beta particles emitted from the sample transfer energy to the solvent
molecules, which in turn transfer their energy to the fluors; the excited
fluor molecules dissipate the energy by emitted light.
● In this way, each beta emission results in a pulse of light.
● Counting efficiencies under ideal conditions range from about 30% for
H-3 (a low-energy beta emitter) to nearly 100% for P-32, a
high-energy beta emitter.

35. The counter has two
photomultiplier tubes
connected in a coincidence
circuit. The coincidence
circuit assures that genuine
light pulses, which reach
both photomultiplier tubes,
are counted, while other
pulses (due to noise, for
example), which would only
affect one of the tubes, are
ignored.

37. Summary of Scintillation Counter
● Scintillation Cocktail contains solvent and fluor (or solute)
molecules.
● Solvents is good at capturing energy of β-particle (electron), but
often does not produce light.
● A fluor molecule enters an excited state following interaction with
excited solvent.
● The excited fluor molecule decays to ground state by emitting light
(usually in blue wavelength)
● Blue light is detected by photomultiplier tube (usually two PMT) are
used to minimise PMT errors.

39. Good radiopharmaceutical requirements :
Since radiopharmaceuticals are administered to humans, they should
posses some important characteristics. The ideal characteristics for
diagnostic radiopharmaceuticals are :
The radiopharmaceutical should be easily produced, inexpensive and
readily available in any nuclear medicine facility. Complicated methods of
production of radionuclide or labeled compounds increase the cost of
radionuclide or labeled compounds increase the cost of the
radiopharmaceuticals.
1. Ease of availability :

40. 2. Short effective half - life
A radionuclide decays with a definite physical half-life. The physical
half-life is independent of any physiochemical condition and is
characteristic for a given radionuclide. Radiopharmaceuticals
administered to human disappear from the biologic system through
fecal or urinary excretion, perspiration or other mechanisms. This
biologic disappearance of a radiopharmaceutical follows an
exponential law similar to that of radionuclide decay. Thus, every
radiopharmaceutical has a biological half-life.

41. 3. No particle emission
Radionuclides decaying by alpha or beta particle emission should
not be used in labeling of radiopharmaceuticals. These particles
cause more radiation damage to the tissue than y-rays do.
Although Y-ray emission is preferable, many Beta emitting
radionuclides, such as 131 I-iodinated compounds, are often used
for clinical therapeutic studies. However, alpha-emitters should
never be used for in-vivo clinical studies because they give a high
radiation dose to the patient.

42. 4. Decay by electron capture or isomeric transition :
Because radionuclides emitting particles are less desirable, the
radionuclides used should decay by electron capture or isomeric
transition without any internal conversion. Whatever the mode of
decay, the radionuclide must emit y-radiation with energy between
30 and 300 KeV. Below 30 KeV, y-rays are absorbed by tissue and
are not detected by the Nal (TI) detector. Above 300 KeV, effective
collimator of y-rays can not be achieved with lead or denser metal.

43. 5. High target to non - target activity ratio :
For any diagnostic study, it is desirable that the
radiopharmaceutical must be localized preferentially in the
organ under study since the activity from non-target areas
can obscure the structural details of the picture of the target
organ. Therefore, the target to non-target ratio should be
high.

44. Radionuclide generator - 99m Tc generator

45. • Tc 99m is the most commonly used radionuclide (80%) Tc is the chemical symbol of
technetium. 99 is its mass number. The m denotes 'metastable',
• Technetium, which is not found in nature, was first discovered by Perrier and Segre in
1937 in a sample of molybdenum that had been irradiated in the Berkeley cyclotron.
• It is useful for several reasons:
• It can be easily combined with several pharmaceuticals.
• Its half-life of six hours is long enough to allow practical imaging but not so long that
the patient, public and environment are over-burdened with radiation.
• It gives off gamma rays at 140keV which is a good match to the sensitivity range of
the Gamma Camera.
• It is a pure gamma emitter.
Technetium 99m

46. a typical of 99m Tc generator

47. Components of generator

48. How does this work
● Saline is initially introduced into the system
● The saline passes through the Molly/Aluminum core
● 99mTc is liberated, in a saline solution while 99Mo remaining in the column
● 99mTc then passes through the Alumina and the glass frit (filter) and then out to elution vial
● The elution vial must be negatively pressured in order for the saline to move through the system
● The type of generator being discussed is known as a dry column generator. Consider that once
the saline passes through the column the remaining air that passes through dries the column as
its pulled through the system. An additional elution vial can be added to further dry the column
● Wet column generated have also been developed
I. This system has a saline reservoir built into the unit
II. The disadvantage to this type of system is that the water remaining in the column can undergo
hydrolysis (converting H2O to H2O2).
III. The advantage to dry column is reduced radiation exposure

49. Application of radionuclides in diagnostics and medical treatment gave start to nuclear
medicine.
It is used primarily to locate tumors in the body, monitor cardiac function following heart
attacks, map blood flow in the brain surgery.
The influence of the ionizing radiation on the biological objects led to the modern technology
that allows physicians to irradiate only selected cells of tumor instead of the entire area.
The advantage of the radionuclide therapy is the absorption of radiation by pathological
centers so that the sound tissues remain intact.
Demand for Purity
Medical radionuclides have to have high radiochemical purity, which demands complicated
radiochemical technology.
Technetium 99m Applications

50. Quality control of Radiopharmaceuticals

51. Quality control of radiopharmaceuticals
Since radiopharmaceuticals are intended to use for human
administration, it is imperative that they undergo strict quality control
measures. The quality control tests are carried out as the following:
physicochemical tests and biological tests.
The physicochemical tests indicate the level of radionuclidic and
radiochemical impurities. However, the biological tests are carried out
essentially to establish the biodistribution, sterility, a pyrogenicity, and
undue toxicity of the radiopharmaceuticals before human administration.

52. Physicochemical tests :
Various in-vitro physiochemical tests are essential for the determination of the
purity & integrity of any pharmaceutical preparation. Some of these tests are
unique for radiopharmaceuticals because they contain radionuclides and are not
applicable to conventional drugs.
The physical appearance, color and state of radiopharmaceutical is important
both on receipt and subsequently. A true solution should not contain any
particulate matter. Colloidal or aggregate preparations must have a proper size
range of particles for a given purpose. In aggregate preparations, the particle size
should vary between 10 to 100 μm
Physical characteristics :

53. pH and ionic strength :
All radiopharmaceuticals should have an appropriate pH value for their
stability and integrity. The ideal pH of a radiopharmaceutical should be
7.4 (pH of the blood), although it can vary between 2 and 9 because of
the high buffer capacity of the blood.
Radiopharmaceuticals must also have proper ionic strength, isotonicity
and osmolarity in order to be suitable for human administration. lonic
strength and pH are important factors for stability of
radiopharmaceutical.

54. Determination of radionuclide purity
The radionucledic purity is defined as the fraction of the total radioactivity in the
form of the desired radionuclide present in a radiopharmaceutical form.
Impuriries arise from extraneous nuclear reactions aree due to the isotopic
impurities in the target material.
The presence of these extraneous radionuclides increases undue radiation
exposure to the patient and may also obscure the scintigraphic imaging.
These impurities can be removed by appropriate chemical methods.
Radionuclidic purity is determined by measuring the half-lives and the
characteristics of the emitted radiations by the individual radionuclides using
multichannel analyzer.

55. Radiochemical purity
● The fraction of the total radioactivity in the desired chemical form in the
radiopharmaceutical
● Radiochemical purity is important in radiopharmacy since it is the
radiochemical form which determines the biodistribution of the
radiopharmaceutical. Radiochemical impurities will have different patterns
of biodistribution which may obscure the diagnostic image obtained and
render the investgation meaningless.
● The presence of radiochemical impurities in a radiopharmaceutical results
in poor-quality images due to the high background from the surrounding
tissues and the blood, and gives unnecessary radiation dose to the
patient.

56. Radiochemical impurities arises from :-
1. Decomposition due to action of solvent
2. Change in temperature or pH of light
3. Presence of oxidising or reducing agent
▫Decomposition of labelled compound by radiolysis depends on -
1. The specific activity of radioactive material.
2. The type and energy of the emitted radiation.
3. The half- life of radionuclide.

57. Analytical methods used to detect & determine the
radiochemical impurities in a given pharmaceutical :
1. Precipitation
2. Paper and instant thin - layer chromatography.
3. Gel chromatography
4. Paper or polyacrylamide Gel electrophoresis.
5. Ion exchange
6. Solvent extraction.
7. High performance liquid chromatography.
8. Distillation

58. Chemical purity
The fraction of the material in the desired chemical
form whether or not all of it is in the labeled form
Aluminum is a chemical impurity in the 99mTc-eluate
The presence of a slight amount of globulins in the
preparation of albumin is indicative of impurities

59. The presence of chemical impurities before
radiolabeling may results in
1. Undesirable labeled molecules that may or may not
interfere with the diagnostic test
2. Undue chemical impurities may also cause a toxic effect

60. Purification of radiopharmaceuticals from
Chemical impurities is often carried out by
methods of chemical separation such as
1. Precipitation
2. Solvent extraction
3. Ion exchange
4. Distillation

61. Biological tests
Carried out essentially to examine
1. Sterility
2. Apyrogenicity
3. Toxicity
Of radiopharmaceuticals before human administration.

62. 1.Sterility
Sterilization indicates the absence of any viable bacteria or
microorganisms in a radiopharmaceutical preparation.
Methods of sterilization
1. Autoclaving
2. Membrane filtration

63. 2. Apyrogenicity
● All radiopharmaceuticals for human administration are required to be pyrogen
free.
● Pyrogens are either polysaccharides or proteins produced by the metabolisms
of microorganisms.
● They are 0.05 to 1mm in size, soluble and heat stable.
● Following administration pyrogens produce symptoms of -
▫Fever, chills, lukopenia, pain in joints, malaise, sweating, headache flushing,
and dilation of pupils.

64. Pyrogenicity Testing
● The principle of the test is based on the formation of an opaque by
pyrogens. upon incubating the sample with LAL at 37 C
● The reaction takes place within 15 to 60 min after mixing and
depends on the concentration of pyrogens.
1. USP Rabbit Test
2. LAL (limulus amebocyte lysate) Test

65. Record Keeping
Record Keeping is mandatory for legal reasons as well as for
tracing any faulty preparations.

66. Radiochemical methods in analysis :
• Radio metric dilution
• Isotope Dilution Analysis
• Activation analysis

67. Radio metric dilution
• One of the oldest methods used for the quantitation of drug molecules is
radiometric analysis. This generally involves quantitation of radiation from
beta-emitting radioactive isotopes such as 14C, 3H or 32P.
• Precise
• Sensitive
• Efficient Detection Method
• It begins with the incorporation into a drug molecule via a chemical synthesis
process.
• Generally a specific carbon atom is replaced with 14C, or a specific hydrogen
atom is replaced with 3H.

68. Advantages
● Detection can be done for multiple matrices
● Method is rapid
● Precise
● The great advantage of this method is that after spike & sample
have been mixed, loss in substance has no longer has an effect
on the result of the analysis

69. Isotope dilution Analysis
▫Isotope dilution analysis is a method of determining the quantity of
chemical substances.
▫ It comprises the addition of known amounts of isotopically- enriched
substance to the analysed sample.
▫ Mixing of the isotopic standard with the sample effectively "dilutes" the
isotopic enrichment of the standard and this forms the basis for the isotope
dilution method.
▫ It is one of the radio activity measurement technique in which the analysis
for sample for which no quantitative isolation is known.
▫ The amount of an inactive element is measured in an unknown mixture.

70. Contd.
▫ Let the inactive element is P and the radio active element is p*. A
known weight (W1) of P tagged with P* is added to the mixture to be
analyzed and its specific activity A1 is known.
▫ The specific activity of a Pure compound (A) is measured.
▫ This method is useful in the analysis of complex biochemical
mixture.

71. Activation Analysis
• Activation analysis is one of the most sensitive and versatile techniques
possible for elemental analysis.
• The technique involves irradiation of a sample with neutrons, charged particles
or photons to induce instability in some of the sample atoms.
• This excitation results in emission of characteristic gamma rays. From the
characteristic rays the nuclei is identified.
• The whole process include three steps :
▫ Activation
▫ Isolation of nuclide
▫ Quantification

72. RADIOIMMUNOSSAY

73. Introduction
When radioisotopes instead of enzymes are used as labels to be
conjugated with antigens or antibodies, the technique of detection
of the antigen-antibody complex is called radioimmunoassay (RIA).
Radioimmunoassay (RIA) is an in vitro assay that measures the
presence of an antigen with very high sensitivity. RIA was first
described in 1960 for the measurement of endogenous plasma
insulin by Solomon Berson and Rosalyn Yalow of the Veterans
Administration Hospital in New York.

75. The classical RIA methods are based on the principle of
competitive binding. In this method, an unlabeled antigen
competes with a radiolabeled antigen for binding to an antibody
with the appropriate specificity. Thus, when mixtures of
radiolabeled and unlabeled antigen are incubated with the
corresponding antibody, the amount of free (not bound to
antibody) radiolabeled antigen is directly proportional to the
quantity of unlabeled antigen in the mixture.

76. Principle of Radioimmunossay
It involves a combination of three principles.
1) An immune reaction i.e. antigen, antibody binding.
2) competitive binding or competitive displacement reaction. (It gives
specificity)
3) Measurement of radio emission. (It gives sensitivity)

77. Immune Reaction
When a foreign biological substance enters into the body bloodstream
through a non-oral route, the body recognizes the specific chemistry
on the surface of foreign substance as antigen and produces specific
antibodies against the antigen so as nullify the effects and keep the
body safe. The antibodies are produced by the body’s immune system
so, it is an immune reaction. Here the antibodies or antigens bind
move due to chemical influence. This is different from principle of
electrophoresis where proteins are separated due to charge.

78. Competitive binding or competitive displacement reaction:
This is a phenomenon wherein when there are two antigens that
can bind to the same antibody, the antigen with more
concentration binds extensively with the limited antibody
displacing others. So here in the experiment, a radiolabelled
antigen is allowed to bind to high-affinity antibody. Then when
the patient serum is added unlabeled antigens in it start binding
to the antibody displacing the labeled antigen.

79. Uses of Radioimmunossay
❏ The test can be used to determine very small quantities (e.g.
nanogram) of antigens and antibodies in the serum.
❏ The test is used for quantitation of hormones, drugs, HBsAg, and
other viral antigens.
❏ Analyze nanomolar and picomolar concentrations of hormones in
biological fluids.

80. The limitations of Radioimmunossay includes :
❏ The cost of equipment and reagents
❏ Short shelf-life of radiolabeled compounds
❏ The problems associated with the disposal of radioactive
waste.

81. References
1. https://chem.libretexts.org/Courses/Northeastern_University/13%3A_Kinetic_Methods/13.3%3A_Radioche
mistryhttps://link.springer.com/chapter/10.1007%2F978-1-4757-4027-1_8
2. https://ehs.princeton.edu/laboratory-research/radiation-safety/radioactive-materials/handling-radioactive-mat
erials-safely
3. https://www.britannica.com/science/radiochemical-analysis#:~:text=During%20use%20of%20the%20radioc
hemical,emissions%20is%20used%20for%20qualitative%E2%80%A6
4. https://www.antibodies-online.com/resources/17/1215/radioimmunoassay-ria/
5. https://humanhealth.iaea.org/HHW/Radiopharmacy/VirRad/Quality_Control_Procedures/Quality_Control_M
odule/Radionuclide_Purity/index.html#:~:text=Radionuclidic%20purity%20is%20defined%20as,total%20radi
oactivity%20of%20the%20source%22.
6. https://www.studyandscore.com/studymaterial-detail/geiger-muller-counter-construction-principle-working-pl
ateau-graph-and-applications
7. https://www.perkinelmer.com/uk/lab-products-and-services/application-support-knowledgebase/radiometric/l
iquid-scintillation-counting.html