Immunological Assay Principles
On this page
Direct answer
Antigen glued to plastic, patient serum added, enzyme-tagged second antibody layered on, substrate colour read — that is an ELISA in one line, and its four formats answer most questions: direct, indirect for antibody, sandwich (two antibodies capturing antigen, the most sensitive for large antigens) and competitive for small antigens like hormones. Agglutination puts the same immunochemistry on visible particles — latex beads for cryptococcal antigen, the Widal tube test for typhoid, Weil–Felix with Proteus strains for rickettsiae — while the Coombs test detects incomplete antibody on or against red cells, and the Mantoux test measures type IV cell-mediated reactivity as induration at 48–72 hours. Western blot, the historic HIV confirmatory test, has been retired in India's NACO algorithm in favour of three-test serial rapid assays.
What you must remember
- ELISA formats: indirect for antibody screening; sandwich (capture) for antigen detection — two distinct antibodies; competitive for small haptens; IgM capture format to dodge rheumatoid-factor interference.
- Rapid platforms: immunochromatographic card tests (dengue NS1/IgM, scrub typhus IgM) trade some sensitivity for bedside speed.
- Widal test: tube agglutination against TO and TH antigens (and AH, BH for paratyphi); in endemic India a single titre of TO ≥1:80 or TH ≥1:160 is conventionally significant, but a four-fold rise in paired sera is far more reliable; antibodies appear only after a week of illness — both false certainty and false dismissal are exam themes.
- Weil–Felix reaction: agglutination of Proteus OX19 (epidemic typhus, Rocky Mountain spotted fever), OX2 (some spotted fevers) and OXK (scrub typhus) — insensitive legacy serology now replaced by IgM ELISA/ICT.
- Coombs test: direct antiglobulin test detects antibody already coating the patient's red cells (haemolytic disease of the newborn, autoimmune haemolysis); indirect test detects free antibody in serum (antenatal Rh screening, crossmatching).
- Mantoux: 5 tuberculin units intradermally; read the induration, not erythema, at 48–72 hours; ≥10 mm is significant in India; positive means infection, not disease — and national policy treats latent infection only in selective high-risk groups.
- Interferon-gamma release assays (QuantiFERON, T-SPOT) use ESAT-6 and CFP10 antigens absent from BCG, so BCG vaccination does not cause IGRA positivity — the preferred test in BCG-vaccinated adults.
- Other classics: Elek immunodiffusion for diphtheria toxin; latex agglutination for cryptococcal antigen; indirect fluorescent antibody tests; neutralisation as the most specific serological correlate of protection; Paul–Bunnell heterophile agglutination in mononucleosis.
Working through a Widal report
A 24-year-old has eight days of fever; the report shows TH 1:160 and TO 1:80. In an endemic country, baseline titres from past exposure and even typhoid vaccination inflate single readings, so this report "supports but does not prove" typhoid — the correct option wording in many screening questions. Confirm with a convalescent sample looking for a four-fold rise, or better, a blood culture in the first week, which remains diagnostic gold. If the same patient has already taken three days of antibiotics, culture yield falls and the Widal becomes a crutch — this is precisely when the exam expects you to argue for culture over serology. Contrast the Weil–Felix scenario: fever with an eschar in the monsoon; OXK weakly positive is a poor performer, so order scrub typhus IgM and start doxycycline empirically.
Where students slip
The direct/indirect Coombs split trips most candidates: antibody on the cell = direct; antibody in the serum = indirect. Mantoux questions punish those who measure erythema or read at 24 hours. And the single most quotable line for India — "IGRA is not affected by BCG" — regularly appears with a distractor claiming Mantoux is superior in the vaccinated.
Frequently asked questions
Which ELISA format is most sensitive for detecting antigen?
The sandwich format, where a capture antibody immobilises the antigen and a second enzyme-labelled antibody detects a different epitope.
What Widal titres are conventionally significant in India?
A single titre of TO at 1:80 or above, or TH at 1:160 or above by the tube method in an endemic setting, with a four-fold rise on repeat testing providing stronger evidence.
How do the direct and indirect Coombs tests differ?
The direct test detects antibody or complement coating the patient's red cells in vivo (as in neonatal haemolysis); the indirect test detects unbound antibody in serum (antenatal screening).
Why is IGRA preferred over Mantoux in BCG-vaccinated adults?
IGRA uses antigens (ESAT-6, CFP-10) absent from BCG and most environmental mycobacteria, so vaccination does not cause false positivity, unlike tuberculin testing.
Which Proteus antigen agglutinates in scrub typhus?
OXK — reflecting cross-reacting antibodies to Orientia tsutsugamushi, though modern IgM ELISA is the recommended test.