Unit 3: Radioisotopes and immunoassay
Clinical Biochemistry-1 notes · PTU syllabus (BMLS403-18)
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Unit summary
Radioisotopes and immunoassays measure tiny amounts of hormones, drugs and antigens. This unit covers instruments for detecting radioactivity, applications of radioisotopes in clinical biochemistry, and ELISA.
After this unit you can
- Describe radioactivity and its units
- Describe instruments for detecting radioactivity
- Explain radioimmunoassay and other uses of radioisotopes
- Explain the principle, types and applications of ELISA
PTU syllabus topics
- Instruments for detection of radioactivity
- applications of radioisotopes in clinical biochemistry
- enzyme-linked immunosorbent assay (ELISA)
- 1
Coat the well with capture antibody
- 2
Add patient sample
Antigen binds
- 3
Wash
- 4
Add enzyme-linked detection antibody
- 5
Wash
- 6
Add substrate
Colour develops
- 7
Read absorbance
Colour ∝ antigen amount
Topic 1
Radioactivity and radioisotopes
- Isotopes have the same atomic number but different mass numbers; radioisotopes are unstable and emit α particles, β particles or γ rays. Half-life: time for half the atoms to decay — ¹²⁵I 60 days, ³H 12.3 years, ¹⁴C 5,730 years, ⁹⁹ᵐTc 6 hours.
- Becquerel (Bq)
- One disintegration per second (SI)
- Curie (Ci)
- 3.7 × 10¹⁰ disintegrations per second
- Gray and sievert
- Absorbed and equivalent dose
Topic 2
Instruments for detecting radioactivity
Geiger–Müller counter
Radiation ionises gas; each event produces a pulse
Surveys for contamination
Gamma (solid scintillation) counter
γ rays produce light flashes in a sodium iodide crystal; photomultiplier counts them
¹²⁵I in radioimmunoassay
Liquid scintillation counter
β particles excite a scintillator cocktail
³H and ¹⁴C
Film badges and TLD
Personal dosimeters
Monitoring staff exposure
Topic 3
Applications of radioisotopes
- Radioimmunoassay (RIA)
- Measuring hormones (T3, T4, TSH, insulin, cortisol), drugs
- Tracer studies
- Metabolic pathways with ¹⁴C and ³H
- Diagnosis
- Thyroid uptake (¹³¹I), bone and organ scans (⁹⁹ᵐTc), ¹⁴C-urea breath test for H. pylori
- Therapy
- ¹³¹I for hyperthyroidism and thyroid cancer
- 1Fixed amount of antibody and radiolabelled antigen
- 2Patient's unlabelled antigen competes for antibody sites
- 3Separate bound from free antigen
- 4Count radioactivity of the bound fraction
- 5More patient antigen → less labelled antigen bound; read from a standard curve
- Safety: ALARA principle (as low as reasonably achievable) — time, distance, shielding; licences from the AERB; radioactive waste rules. Non-radioactive immunoassays have largely replaced RIA.
Topic 4
ELISA
- Enzyme-linked immunosorbent assay: an antigen–antibody reaction detected by an enzyme label (horseradish peroxidase or alkaline phosphatase) acting on a substrate (TMB, OPD) to give a colour measured at 450 nm.
Direct
Enzyme-labelled antibody binds the coated antigen
Simple antigen detection
Indirect
Patient antibody binds coated antigen; enzyme-labelled anti-human antibody detects it
Antibody detection — HIV, hepatitis C, dengue IgG
Sandwich
Capture antibody binds antigen; second enzyme-labelled antibody binds it
Antigen detection — HBsAg, hormones, NS1
Competitive
Sample antigen competes with labelled antigen
Small molecules — drugs, T4
- 1Coat wells with antigen; block free sites
- 2Add patient serum; incubate; wash
- 3Add enzyme-labelled anti-human IgG; incubate; wash
- 4Add substrate; colour develops
- 5Stop with acid; read absorbance; compare with cut-off
- Advantages: sensitive, no radiation, automated, many samples. Variants: chemiluminescence immunoassay (CLIA) and ELFA give even higher sensitivity.
Key terms
- Half-life
- Time for half of a radioisotope to decay
- Becquerel
- SI unit of radioactivity
- Scintillation counter
- Detector counting light flashes from radiation
- Radioimmunoassay
- Immunoassay using radiolabelled antigen
- ELISA
- Immunoassay using an enzyme label and colour reaction
Quick revision
- Isotopes, α, β, γ; half-life examples; Bq, Ci, Gy, Sv.
- GM counter, gamma counter, liquid scintillation, dosimeters.
- RIA principle; diagnostic and therapeutic uses; ALARA, AERB.
- Direct, indirect, sandwich, competitive ELISA; steps; CLIA.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Define half-life.
- Q2.What is a becquerel?
- Q3.Which counter is used for ¹²⁵I?
- Q4.State the principle of competitive RIA.
- Q5.Which ELISA detects HBsAg?
- Q6.Name two enzymes used as ELISA labels.
Long-answer questions
- Q1.Describe instruments used for detecting radioactivity.
- Q2.Explain radioimmunoassay and the applications of radioisotopes.
- Q3.Explain the principle, types and applications of ELISA.
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