Immunology and Serology Testing

An antigen is a substance (usually a protein or carbohydrate) that triggers an immune response. It can be from a pathogen (bacterial cell wall protein, viral coat protein), an allergen (pollen protein), or the body's own proteins (in autoimmune disease). Antigens are recognized as 'foreign' or 'abnormal' by the immune system. An antibody (immunoglobulin) is a Y-shaped protein produced by B cells in response to an antigen. Antibodies bind to antigens with high specificity (like a lock and key) and are the immune system's weapon: they tag pathogens for destruction, neutralize toxins, and activate complement. Serologic testing detects either the antigen (pathogen presence) or the antibody (immune response to the antigen). Example: In HIV testing, we test for HIV antigen (p24 protein) to detect acute infection, or for anti-HIV antibodies to detect immunity. In bacterial infection, we culture the pathogen (antigen) or test for antibodies (immune response). The test chosen depends on the stage of infection: early = test for antigen; later = test for antibodies.

ELISA (Enzyme-Linked Immunosorbent Assay) is the most common immunologic assay in clinical labs. It detects antigens or antibodies by coupling the immune reaction to an enzyme that produces a color change. Sandwich ELISA (detects antigen): (1) Coat a plate well with capture antibody (specific to the target antigen). (2) Add patient serum. If the antigen is present, it binds to the capture antibody. (3) Add detection antibody (another antibody to the antigen, linked to an enzyme like HRP). (4) Add substrate (like TMB), which the enzyme converts to a colored product (blue or yellow). (5) Measure the color intensity on a plate reader; intensity = amount of antigen. Indirect ELISA (detects antibody): (1) Coat plate with antigen. (2) Add patient serum. If antibodies to the antigen are present, they bind. (3) Add anti-human IgG (antibody to human antibodies) linked to an enzyme. (4) Add substrate and measure. Uses: HIV testing (detects anti-HIV antibodies), hepatitis C (anti-HCV), pregnancy (hCG antigen), allergy testing (specific IgE). Advantages: sensitive, specific, quantitative (can measure amount of antigen/antibody), and automated. Disadvantages: takes time (hours to overnight), requires careful technique, and reagents must be kept cold.

The ANA test detects antibodies against components of the cell nucleus (DNA, histones, other nuclear proteins) and is a screening test for autoimmune diseases, especially systemic lupus erythematosus (SLE). ANA is positive in ~95% of SLE patients, making it highly sensitive for SLE screening. However, ANA is not specific: it is also positive in other autoimmune diseases (rheumatoid arthritis, scleroderma, Sjögren's syndrome, antiphospholipid syndrome) and in healthy people (~5% of healthy controls). Interpretation: ANA-negative essentially rules out SLE; ANA-positive requires follow-up. If ANA is positive, reflex to specific antibody testing: anti-dsDNA (double-stranded DNA) = specific for SLE; anti-Sm (Smith antigen) = specific for SLE; anti-histone = drug-induced lupus; anti-Ro/La = Sjögren's syndrome, neonatal lupus; anti-centromere = limited scleroderma; anti-Scl-70 = diffuse scleroderma. ANA patterns (homogeneous, speckled, nucleolar, centromere) are less specific than specific antibody tests but can provide clinical clues. A positive ANA with high-titer anti-dsDNA and anti-Sm in the context of clinical symptoms (rash, photosensitivity, arthritis, fever, kidney involvement) = diagnosis of SLE.

Seroconversion is the point in time when a patient's serum changes from negative to positive for a specific antibody or antigen. It occurs when a person is infected and the immune system generates a response. The timing varies by infection: in HIV, seroconversion occurs 2–12 weeks after infection (window period where tests may be negative even though the person is infected and contagious); in hepatitis B, anti-HBc (core antibody) appears first (4–6 weeks), then HBsAg (surface antigen), then anti-HBs (surface antibody, protective immunity). In acute infections, IgM antibodies appear first (early response), then IgG (late/long-term immunity). Serologic interpretation by stage: (1) Acute/early infection: antigen (HBsAg, HCV antigen, HIV p24) and/or IgM antibody positive; IgG negative. (2) Convalescent/recovery: IgM declining, IgG rising. (3) Chronic/past infection: IgG positive, IgM negative (unless reactivation). (4) Immunity: specific protective antibody present, no antigen. Testing during the window period (before seroconversion) can produce false-negative results, so repeat testing is recommended for high-risk exposures. Understanding seroconversion kinetics prevents misinterpreting negative tests in early infection.

IgM (immunoglobulin M) and IgG (immunoglobulin G) are two types of antibodies produced in response to infection or vaccination. IgM is the first antibody made (appears 1–2 weeks after infection or vaccination), peaks at 1–2 weeks, then declines over 3–6 months. IgM indicates acute or recent infection. IgG appears later (2–4 weeks), peaks over weeks to months, and persists for years or life. IgG indicates past infection, immunity, or chronic infection. Clinical interpretation: (1) Positive IgM, negative IgG = acute infection (symptoms should be present). (2) Positive IgG, negative IgM = past infection or immunity (no symptoms expected). (3) Positive IgM and IgG = recent infection (in past weeks to months). (4) Negative IgM, positive IgG = past infection (immune), or very early/late in infection (unlikely to have disease). Examples: (1) Acute rubella = IgM positive, IgG negative (live attenuated vaccine changes to IgG positive weeks later). (2) Chronic hepatitis B = HBsAg and anti-HBc positive, IgM anti-HBc negative (indicating chronic, not acute). (3) Toxoplasma serology in pregnancy: IgG positive (past infection, fetus immune unless reactivation), IgM positive (acute infection, risk to fetus). Testing for both IgM and IgG and understanding their kinetics is critical for dating infections and guiding treatment.

A Western blot is a protein immunoassay that separates and identifies specific proteins in a sample using protein denaturation, gel electrophoresis, and antibody detection. Steps: (1) Extract proteins from tissue or serum. (2) Denature proteins (break apart) with heat and reducing agents. (3) Run samples through an SDS-PAGE gel (separates proteins by molecular weight). (4) Transfer proteins to a membrane (blotting). (5) Incubate with primary antibody (targets the protein of interest). (6) Incubate with secondary antibody linked to enzyme or fluorescent label. (7) Develop to visualize protein bands. Each band represents a protein of a specific molecular weight. Uses: (1) Confirmatory HIV test (detects multiple HIV antibodies to different HIV proteins: gp120, gp41, p24). (2) Confirmatory Lyme disease (detects antibodies to multiple Borrelia burgdorferi proteins). (3) Confirmatory hepatitis B core (anti-HBc) and surface (anti-HBs). (4) Research: quantify proteins in tissue samples. Advantages: highly specific (detects individual proteins), can separate multiple proteins. Disadvantages: time-consuming (24–48 hours), requires skilled personnel, expensive. Western blot is often used as a confirmatory test after a screening test (like ELISA) is positive.

Blood typing is the identification of ABO and Rh blood group antigens on red blood cells (RBCs) to ensure safe transfusion. ABO types: Type A (A antigen), Type B (B antigen), Type AB (both A and B), Type O (neither). Rh type: Rh-positive (D antigen present) or Rh-negative (D antigen absent). Naturally occurring antibodies: Type A people have anti-B antibodies; Type B have anti-A; Type AB have neither; Type O have both anti-A and anti-B. If Type A blood is transfused to a Type B person (who has anti-A antibodies), the antibodies attack the RBCs, causing hemolysis and potentially fatal transfusion reaction. Cross-matching tests whether donor RBCs will be attacked by recipient antibodies: (1) Major cross-match: add donor RBCs to recipient serum; if agglutination (clumping), the transfusion is incompatible. (2) Minor cross-match: add recipient RBCs to donor serum; less critical but checks for rare donor antibodies. Rh sensitization: Rh-negative people have no anti-D naturally, but if exposed to Rh-positive blood (transfusion, pregnancy), they develop anti-D antibodies. A second Rh-positive transfusion then causes hemolysis. Prevention: (1) Type and cross-match before every transfusion. (2) In Rh-negative women giving birth to an Rh-positive infant, give anti-D immunoglobulin (RhIG, WinRho) within 72 hours to prevent sensitization. (3) Type and cross-match pregnant women early for alloimmunization screening. The consequences of a hemolytic transfusion reaction (fever, chills, hemoglobinuria, renal failure, shock, death) make accurate typing and cross-matching life-saving.