RIARadioimmunoassay

Remember Immunoassay reactions may be

competitive or non-competitiveCompetitive labeled known and patient unknown are added to

reaction and “compete” for the target.Non-competitive–Add patient sample, for example looking for

antibody, to known reagent antigen.–Reaction occurs and the concentration is

directly related to the amount of antibody in patient sample.

Definition

Radioimmunoassay (RIA) is a very sensitive Competitive binding assay used to measure concentrations of antigens (for example, hormone levels in blood) using the specificity of antigen – antibody binding and quantitation using radioactivity.

Radioactive isotopes are usually high specific activity H3 (beta) , I125 (gamma).

This technique used for detection of micro-quantites of proteins, hormones, viral antigens, antibodies, vitamins and drugs.

RIA is used in various branches of science like Biochemistry, microbiology, Hematology, endocrinology and Clinical Pharmacology.

Highly specific (antibody dependent)

One of the most sensitive techniques for detecting

antigen or antibody, it is possible to detect a few picograms (10^-12 g) of hormone in the tube.

Easy signal detection Well-established, rapid assays

Advantages of RIA

Disadvantages 1. Hazardous due to use of radioactivity 2. The time and expense associated with

maintaining a licensed radiation safety and disposal program.

3. The health and safety risks posed by the use of radiation

4. Need expensive equipments as γ or β counter & Radiolabeled Isotopes

5. The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4) synthesis result in hypothyroidismFor these reasons, RIA has largely

replaced in routine clinical laboratory by enzyme immunoassay.

PrecautionsIt requires special

precautions and licensing, since radioactive substances are used.

1.Pregnant females should not work in an area where RIA tests are being performed.

2. Personnel handling isotope reagents must wear lead-coated gown & gloves to limit their exposure to radiation.

3. Special sinks and waste disposal containers are required for disposal of radioactive waste.

4. The amount of radioisotope discarded must be documented for both liquid and solid waste.

5. Leakage or spills of radioactive reagents must be measured for radioactivity; the amount of radiation and containment and disposal processes must be documented.

6. The work must be in a lab with lead-covered floor or another radioabsorbent leather.

7. Radioisotope waste should be disposed in a special way.

Principle of RIA Based on competition

between unlabelled antigen and finite amount of corresponding labeled antigen usually with Iodine 125 for a limited number of high-affinity antibody binding sites in a fixed amount of antiserum.

The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody.

As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites. The amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured

in order to determine the amount of antigen present in the test sample.Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ag*

Relationship between radiolabeled Ag and unlabeled (serum’s) Ag

RIA Kit1. Ab-coated plastic tubes

in addition to Total count tube Non-specific Binding tube2. Calibrators with known

cons. of unlabeled Ag3. Sample Diluents

4. I125 ﴾ radio-labeled Ag﴿5. Washing buffer

Mark all the tubes

Assay Procedure

Total count tube

Non-specific binding tube

Sample/s

Addition of samples and calibrators ( unlabeled Ag )to their corresponding tubes.

Calibrators

5 10

15

20

25

0

Contain labeled Ag only

Addition of labeled Ag ( I125 ) to all the previous tubes

Incubation at room temperature.Centrifugation for 15 min. at 1500

rpm.Decant & wash contents of the tubes except Total tube

The radioactivity of each is measured by Gamma counter

Count Gamma emission Counts per minute (CPM) for each tube A sample containing a higher concentration of the unknown antigen will have a lower CPM

γ-counter

From these data, a standard binding curve, like the one shown in red, can be drawn.

CPM count expresses the labeled Ag (I125) conc.

As seen in the diagram, radioactive signal of I125 is inversely proportional to unlabeled Ag conc. in sample.

Important points Total binding tube act as a control, neither

washed nor decanted. Non-specific binding tube (NSB) contain only

labeled Ag I125 , used to exclude any residual free iodine after decant step.

Zero calibrator tube shows maximum binding, there is no Ag to compete with the labeled Ag for Ab binding sites.

The competition increases as the concentration of serum Ag increases, and so I125 binding decreases

As CPM are high numbers, We can

construct another curve between Ag concentration and percentage of CPM of each tube using this formula :

%S = CPM S * 100%CPM MB

S= sampleMB = maximum binding tube

Unlabeled Ag conc.

perc

enta

ge

An example of A drug determination by RIA immunoassay

IRMA

Immunoradiometric assay

(Non competitive)

Immunoradiometric assay (IRMA)

The IRMA is a noncompetitive assay in which the analyte to be measured is 'sandwiched' between two antibodies. The first antibody is immobilized inside the walls of the tubes.

The other antibody is radiolabeled for detection. The analyte present in the samples, standards

and controls is bound by both antibodies to form a 'sandwich' complex.

Advantages of IRMA include a faster reaction rate and an increased sensitivity because the antibody excess allows all of the unknown analyte to be involved in the reaction.

Principle of IRMA

It resemble the principle of non-competitive Sandwich ELISA but the label here is radioactive I125 rather than enzyme and the method of signal

detection certainly different.

False positive & False negative results

in immunoassaysAlthough immunoassays are both highly

sensitive and specific, false positive and negative results may occur.

 False-negative results from1. improper sample storage or treatment2. reagent deterioration3. improper washing technique.

False-positive results 1. For samples containing small fibrin strands

that adhere to the solid phase matrix.2. By substances in the blood or urine that cross-

react or bind to the antibody used in the test.