Primary Reactions 2.1 RIA

Question 1

____ is an in-vitro technique used to measure concentrations of antigens (for example, hormone levels in the blood) without the need to use bioassay.

Answer 1

Radioimmunoassay (RIA)

Question 2

The three (3) principles of RIA:

Answer 2

a. ) Immune reaction i.e. antigen, antibody binding.
b. ) Competitive binding or competitive displacement reaction. (for specificity)
c. ) Measurement of radio emission (for sensitivity)

Question 3

The Uses of RIA –

Also, to quantitate: _______

Answer 3

● Measure growth of hormone levels 
● For drug/narcotics detection 
● Tracking of Leukemia virus 
● Blood Bank Hepatitis virus screening 
● Peptic ulcer diagnosis & treatment 
● Early cancer detection 
● Research with brain chemicals, i.e. neurotransmitters

HCG, FSH, Gastrin, Insulin, CEA, Thyroxin, TSH, Estrogens, Androgens, Ig

Question 4

Advantages of RIA:
● Immune reactions are highly _____
● Immune reactions are highly ______.
● Technique is extremely sensitive and precise in determining _______
● RIA measures down to the _____ range (10-12g.), any compound to which an antibody can be produced.
● Technique allows accurate quantitation of a wide variety of biologically important compounds, e.g. _______, hormones, vitamins and drugs, in biologic fluids or tissues in very low concentration.
● Can analyze ____ and ____ concentrations of hormones in biological fluids.
● The decay process of radioisotopes is not affected by ________, such as pH, ionic strength, and inhibitors dependent on optical measurements or enzymatic activities.

Answer 4

specific. 
sensitive
trace amounts of analytes that are small in size.
picogram
peptides
nanomolar & picomolar
factors common to biological media

Question 5

➢ Disadvantages of RIA –
● _____: The technique uses radiolabeled reagents.
● Requires specially __________.
● Laboratories require special license to handle radioactive material.
● Requires special arrangements for –
○ Requisition and storage of radioactive material.
○ Radioactive waste disposal.
● Some radiolabeled compounds are relatively unstable, and have ______.
● Both _______ equipment and reagents are expensive.

Answer 5

Radiation hazards
trained professionals
short half-life
gamma & beta Spectrometer

Question 6

______is the form of decay, measured most commonly in RIA.

Answer 6

Gamma ray emission

Question 7

Radioactive Labels used in RIA:

Answer 7

131l
125l
3H

Question 8

Radioactive Labels used in RIA:

It has a half-life of 60 days; can be easily incorporated into protein molecules; emits gamma rays which is detected by a gamma counter

Answer 8

125I

Question 9

Radioactive Labels used in RIA:

It is gamma-emitting isotope; has half-life of only 8.05 days.

Answer 9

131I

Question 10

Radioactive Labels used in RIA:

It is beta-emitting isotope

Answer 10

3H - (Tritiated Hydrogen)

Question 11

Measuring Radioactivity:

● Measured on either the_____, the ___ or both.
● The _________ – detects & measures products of radioactive decay,
a.___________ – the high energy gamma rays from 125I or 131I.
b. __________ – the beta-emitting isotopes.

Answer 11

supernatant fluid, precipitate

Scintillation Counter

a. Crystal Scintillation Counter
b. Liquid Scintillation Counter

Question 12

This has higher detection efficiency

Answer 12

Liquid Scintillation Counter

Question 13

________detects & measures products of radioactive decay,

Answer 13

The Scintillation Counter

Question 14

An unknown quantity of antigen in patient’s serum is allowed to compete for antibody binding with a known quantity of the same antigen to which a radioactive isotope (tracer) is covalently bonded

Answer 14

Principle of RIA:

During incubation, the labelled and unlabeled antigens compete for a limited number of antibody combining sites.

Question 15

Principle of RIA:

“If unlabeled antigen is in higher concentration than the labeled antigen, more antigen-antibody complexes will ______. The greater the radioactivity, the ____ would be the concentration of antigen present in the sample.”

Answer 15

not be radioactive
lesser

The radioactivity in the bound phase and/or the free phase is quantitatively measured.

Question 16

RIA techniques commonly employed to measure total serum IgE levels:

Answer 16

1. Indirect Radioimmunosorbent Test
2. Direct Radioimmunosorbent Tes (RIST)
3. Radioimmunoprecipitation Test (RIP)
4. Radioallergosorbent Test (RAST) - Quantitative 5. Immunoradiometric Assay (IRMA)

Question 17

Principle of RIA.

Labeled antigen competes with patient antigen for a limited number of binding sites on solid phase antibody.

(A) Very little patient antigen is present, making radioactivity of the solid phase ____.
(B) More patient antigen is present, and the radioactivity of the solid phase is _____ in proportion to the amount of patient antigen bound.

Answer 17

high

reduced

Question 18

➢ A non-competitive binding assay

Answer 18

RADIOIMMUNOSORBENT TEST (RIST)

Question 19

RADIOIMMUNOSORBENT TEST (RIST) 
Principle: 

● _______is covalently coupled to cross-linked Dextran particles (Sephadex) or solid phase carriers. Patient’s serum (IgE) is added.
● Then, a constant amount of radiolabeled (usually 125I) IgE is added and binds with receptor sites in patient’s IgE in the patient’s serum sample.
● Following incubation, the tubes are centrifuged and washed ____. Decantation after the last wash leaves a pellet of complexes consisting of Sephadex-bead/solid carrier-Anti-IgE- IgE.
● Emission of gamma rays per unit time is counted and a standard curve constructed.

Answer 19

Anti-IgE

(3X) with buffer

Question 20

[RIST]

The higher the concentration of IgE in the patient’s sample, the ____________

Answer 20

higher the number of counts in the pellet.

Question 21

A blood test to detect WHAT allergen or substances a patient is allergic to that is causing the disease, and measure the increase in IgE levels. Paper discs with different antigens on them are used to test for allergy. -

Answer 21

RAST: The RadioAllergoSorbent Test

Question 22

[RAST]
Example: ✓ if patient is allergic to “X” – add the patient’s serum to a paper disc that contains this antigen. If the serum contains antibodies against X then they will bind to each other. These antibodies will, of course, be____

Answer 22

IgE (anti-X).

Question 23

measures allergen-specific IgE activity

Answer 23

Radioallergosorbent test (RAST)

Question 24

➢ Sensitivity is about 1 International Unit IgE/ml.

● 1 IU is equivalent to approximately ___. of IgE protein

Answer 24

RADIOIMMUNOPRECIPITATION ASSAY (RIP)

2.4 ng

Question 25

Principle:
Patient’s serum is mixed first with rabbit antihuman IgE and second with radiolabeled IgE myeloma protein.After incubation, the soluble IgE-anti-IgE complexes are precipitated with goat anti-Rabbit gamma globulin. All tubes are incubated, centrifuged and washed thoroughly to remove unbound label and counted.

Standard reference curve is plotted and IgE concentration is interpolated from the curve

➢ Disadvantage: As competitive binding assay it is _____ to interference by serum factors at high serum concentration.
➢ Advantage: The addition, of a pre-titrated amount of normal Rabbit serum (or purified Rabbit gamma globulin) before the introduction of goat antirabbit gamma globulin, ______ to the formation of larger aggregates, diminishes the loss of soluble complexes, and thereby _____ the sensitivity of the test.

Answer 25

Double Antibody Technique

susceptible
contributes
increases

Question 26

➢ Noncompetitive immunoassay: Uses labeled antibody that is present in excess.

Answer 26

NON-COMPETITIVE IMMUNORADIOMETRIC ASSAY (IRMA)

Question 27

NON-COMPETITIVE IMMUNORADIOMETRIC ASSAY (IRMA)

Principle:
● ________ is removed by the addition of excess analyte coupled to a solid phase.
● The supernatant containing the bound complexes is counted, and the amount of bound labeled antibody is in ____proportion to the amount of patient analyte.

Answer 27

Unreacted antibody

direct

Question 28

NON-COMPETITIVE IMMUNORADIOMETRIC ASSAY (IRMA)

Advantages:

1. ___ reaction rate.
2. _____ because the antibody excess allows all of the unknown analyte to be involved in the reaction.

Disadvantages:

1. There may be ______ because the increase in antibody concentration can result in cross-reactivity with other antigens
2. _____ involved in working with radioactive substances.
3. The need for ____ equipment.

Answer 28

Fast
Increased sensitivity

loss of specificity
Health hazard
expensive

Question 29

IRMA with excess labelled antibody.
✓ Patient antigen is added to a tube with excess labelled antibody.
✓ After incubation, solid-phase antigen is added. This will combine with any labeled antibody that has not bound patient antigen.
✓ The tube is centrifuged, and the solid-phase antigen goes to the ____.
✓ Radioactivity remaining in the ____ is determined.
✓ The count obtained is ______ to the amount of patient antigen present in the specimen.

Answer 29

bottom
supernate
directly proportional