1. RADIOIMMUNOASSAY (RIA)
Asad Vaisi-Raygani
Professor of Clinical Biochemistry

2. INTRODUCTION
RIA is a nuclear technique widely used for measuring minute substances with IN VITRO Procedures.
It combined the technology of nuclear medicine (tracer technique) and immunology (antigen-antibody binding) so that the name RIA is designated. Someone called it was a hybrid technique.

4. In earlier days RIA only limited to certain hormone, but now the scope greatly expanded to the field of reproductive physiology, oncology, immunology, hematology, pharmacology and parasitology etc.
It makes a great contribution to the diagnostic laboratory and scientific research works.

5. Antibody – Antigen Interactions:
IMMUNOASSAYS
Introduction
Antibody – Antigen Interactions:
The body contains between 106 and 108 types of antibodies
Each antibody has the ability to bind to a different foreign agent, or antigen (Ag)
The ability of an antibody to recognize and bind a given antigen depends on the structure of its binding site
Determined by the amino acid sequence of the antibody near the N-terminal ends of the heavy and light chains

6. IMMUNOASSAYS
Introduction
Antibody – Antigen Interactions:
The general reaction between a single binding site on the antibody (Ab) and antigen (Ag) can be written as follows:
where Ka is the binding or association equilibrium constant
The value of Ka is typically in the range of 106 to 1010 M-1
The binding is very selective and only occurs between Ab and Ag, or between Ab and molecules similar to Ag in their three-dimensional structure. Ka
Ab + Ag ↔ Ab-Ag

7. I Introduction Antibody Production – polyclonal antibodies:
If the agent is a foreign to the animal, the animal will develop antibodies to the agent and release these antibodies into its blood.
After a few months, blood is removed from the animal and the antibodies produced are collected for use
Antibodies produced in this fashion are typically very heterogeneous
Recognize a number of different sites on the analyte
Binding with a range of affinities (Ka)
Heterogeneous antibodies are known as polyclonal antibodies
Arise from several different lines of antibody-producing cells within the animal
IMMUNOASSAYS

8. Commercial production of antibodies: polyclonal vs monoclonal
Host animals ca be used to raise antibodies against a given antigen

9. Antibody Production - monoclonal antibodies (mAb):
IMMUNOASSAYS
Antibody Production - monoclonal antibodies (mAb):
Monoclonal antibodies differ from polyclonal antibodies in that they are produced by a single cell line within the body
All monoclonal antibodies from the same cell line recognize the same site on an analyte and bind with an identical binding affinity (Ka)

13. displaces labeled antigen
IMMUNOASSAYS
Competitive binding immunoassays
Quantitative method based on competition between analyte in sample and a fixed amount of labeled analyte for a limited number of antibody binding sites (equilibrium method)
Indirectly measures the amount of analyte in the sample by looking at amount of labeled analyte it displaces from the antibody
Unlabeled
antigen
Unlabeled antigen
displaces labeled antigen

14. Competitive binding immunoassays
Quantitative method based on competition between analyte in sample and a fixed amount of labeled analyte for a limited number of antibodA typical calibration curve for the assay
y binding sites (equilibrium method)
IMMUNOASSAYS
Linear
transform
Ln(antigen concentration)

17. Competitive binding immunoassays
Advantages of competitive binding immunoassay
Can be used with any type of analyte
Good limit of detection
Theoretical limit: 1/Ka or 10-6 to M
Few interference from other compounds in sample

18. Disadvantages of competitive binding immunoassay
Some skill required to obtain optimum conditions for assay
Long incubation times (hours-days)
Limit of detection ultimately controlled by quality of antibody
Antibody binding strength (Ka)
Detection limit varies between different antibody preparations

19. Only useful for large analytes
1000 to 2000 MW
Requires enough room on molecule to bind two antibodies simultaneously
Requires multiple antibodies per analyte
Usually manual method

20. between two antibodies
Sandwich immunoassays
Quantitative method based on use of two antibodies to detect analyte
First antibody extracts analyte from sample
Second antibody (containing chemical label) identifies presence of analyte
IMMUNOASSAYS
Unlabeled
antigen
antigen “sandwiched”
between two antibodies
Solid
support

21. This type of assay measures the amount of analyte in the sample by looking at the amount of labeled antibody that binds to analyte on the solid support

22. Sandwich immunoassays
Quantitative method based on use of two antibodies to detect analyte
A typical calibration curve for the assay
Response
Concentration of Analyte

23. Sandwich immunoassays
Advantages of sandwich immunoassay
Linear calibration curve
Lower limits of detection possible than with competitive binding immunoassay
< M
Greater selectivity than competitive binding assay
Two antibodies instead of one are used to recognize analyte
Shorter incubation times than competitive binding assay (hours vs. days)
Less susceptible to variations in quality of antibody preparation then competitive binding assay

24. Labels for Immunoassays
The selectivity of a competitive binding assay depends on the specificity of the antibody
The use of a chemical label is also required
Several types of chemical labels have been used in immunoassays
Type of Label
Example
Measurement Principal
Limit of Detection
Radiolabels
I125
Radioactive delay
10-13 M
Fluorescent
Fluorescein,
Rhodamine
Fluorescence
10-10 M
Rare earth chelates
Time-resolved fluorescence
Enzymatic
Horse radish peroxidase
Formation of colored product by enzyme
10-11 M
Chemiluminescent
Acridinium esters, luminol
Light production by chemical reaction

26. Separating reagent (SR) Separation of B and F 1
Separating reagent (SR) Separation of B and F 1. Non specific SR: (absorbing F) DCC(), PEG() 2. Specific SR: (absorbing B) second antibody 3. Solid phase SR: (absorbing Ab on solid material)

28. IRMA
Ag+*Ab Ag.*Ab+*Ab
(B) (F)

29. Ag can bind to specific. Ab to form Ag. Ab complex (B), and leave free
Ag can bind to specific *Ab to form Ag*Ab complex (B), and leave free *Ab (F).
The amount of B formed is proportional to the Ag originally present in serum.
Separate B and F.

30. IRMA Ag

31. COMPARE IRMA WITH RIA RIA IRMA Labeled substance Ag Ab
Principle Competing depression Noncompetitive
Ab Limited Overdose
Standard curve Negative Positive
Balance Slow Fast
Measure range Narrow Wide
Measure object Large or small molecular Large molecular

32. QUALITY INDEX High Sensitivity Strong Specificity Precise in Precision
Good Accuracy

33. Non Specific Binding : What is it?
Secondary antibody
Primary Antibody
Antigen
PG electrode
In addition to binding to receptors of interest, sec. antibody may also bind to other sites. Binding to the receptor of interest is called specific binding, while binding to the other sites is called nonspecific binding (NSB).
NSB can be minimized by saturating the unoccupied binding sites with a blocking reagent (NSB agent) without taking active part in specific assay reaction.
Detergent blocking agents are non ionic.

34. Immunoassay without NSB blocking agent
Enzyme labelled
Secondary antibody
Primary Antibody
Antigen
PG electrode t
Enzyme (HRP) labelled sec antibodies are conjugated to antigen. I
Voltage+H2O2
Signal

35. Immunoassay with NSB Blocking agent
Enzyme labeled
Secondary antibody
Primary antibody
Antigen
Blocking agent I t
PG Elecrode
Voltage+H2O2
Signal

36. Properties of the blocking agent.
Inhibit NSB (passive or covalent) of assay components to the surface,
Inhibit non-specific protein - protein interaction.
Exhibit no cross reactivity with subsequent assay components ( antibodies, protein )
Not disrupt the bonds that immobilize the specific protein or biomolecule to the surface.
Exhibit consistent, reproducible performance with every lot.

37. Blocking agents Detergent Blockers Protein Blockers
Tween-20, Triton X-100
Protein Blockers
Bovine serum albumin, Casein, Fish Gelatin, Whole Sera,
Polymer based Blockers
Polyethylene glycol (PEG), Polyvinyl alcohol (PVA), Polyvinylpyrrolidone (PVP), Polyacrylic acid (PAA), Polyacrylic maleic acid (PAMA).

38. PSMA Immunoassay (Prostate Specific Membrane Antigen)
HRP labeled anti-PSMA1
anti-PSMA2
PSMA
Blocking agent I t
SWCNT
PG Electrode
Voltage+H2O2
Signal

42. Problems/difficulties
If your test tube isn’t cleaned then the sample will be contaminated
Plug in wrong data
Properties of specimens difficult to detect
Interference
Malfunction on the instrument used to count the radioactivity
Radioactive isotope Antibodies and antigens aren’t pure