A. Agglutination assays demonstrate the presence of antigen-antibody reactions by the visible aggregation of antigen-antibody complexes. These tests are simple to perform and are often the most sensitive test method.
1. Flocculation tests. Antibody is detected when soluble antigen interacts with antibody and a precipitate is formed. Antigen is bound to reagent particles; visible agglutination results when the particles bind to antibody.
2. Latex agglutination. Antibody is bound to latex beads; visible agglutination occurs when antigen binds to the latex-bound antibody.
3. Direct bacterial agglutination. Antibodies bind to the surface antigens of bacteria in suspension, which results in visible agglutination.
4. Hemagglutination tests are used to detect antibodies to red blood cell (RBC) antigens.
In passive or indirect hemagglutination tests, soluble antigens are adsorbed onto the
surface of RBCs. These antigens then bind to any corresponding antibody present, and the RBCs agglutinate.
5. Agglutination inhibition assays are very sensitive and can detect small amounts of antigen. In hemagglutination inhibition (HAI) assays, antigen and antibody are bound to RBCs. If antibody is present in the test sample, agglutination does not occur because there is already antibody bound to the antigen. Agglutination is a negative result. In other assays, antigen and antibody are bound to latex particles.
B. Precipitation assays. When both are present in proper proportions, antigens and antibodies interact and form visible precipitates. The largest amount of precipitation is seen when antigens and antibodies are present in optimal proportions; this is known as the equivalence zone. False-negative reactions can occur when either antigen or antibody is present in excess.
(Web Color Images 5–12 and 5–13)
1. Double immunodiffusion (Ouchterlony method). Antigens and antibodies are al- lowed to diffuse in a semisolid medium such as agar or agarose. When the antigens and antibodies meet, a precipitate forms. Three basic reaction patterns can result from this interaction (Web Color Image 5–14).
a. Identity. A single smooth arc of precipitation forms between the antigens and antibodies, which indicates that the antibodies are precipitating identical antigen specificities.
b. Nonidentity. Two separate lines of precipitation cross each other, which in- dicates that the antigens and antibodies are unrelated and do not precipitate together.
c. Partial identity. The two precipitating lines meet, forming a spur. This indicates that the antigens share some common epitopes, but that one of the antigens has a unique epitope.
2. Radial immunodiffusion (RID) is a single diffusion method that can be used to quan- titate immunoglobulins and other serum proteins. Samples are introduced into wells cut in agarose containing antiserum and allowed to diffuse, usually overnight. The di- ameters of the resulting precipitation rings correspond to the amount of antigen present (Web Color Image 5–15).
3. Electroimmunodiffusion combines the speed of electrophoresis with the sensitivity of immunodiffusion.
a. Countercurrent immunoelectrophoresis (CIE). This technique is essentially a double diffusion technique in which voltage is applied to move the antigens and antibodies together. A precipitin band forms when a zone of equivalence is reached.
b. Rocket electrophoresis. This technique applies an electrical charge to a RID assay, which results in a rocket-shaped line of precipitation. The height of the rocket is proportional to the antigen concentration.
C. Electrophoresis
1. Immunoelectrophoresis (IEP) is often used to diagnose monoclonal gammopathies.
After the serum or urine specimen is electrophoresed in a gel medium, a trough is cut in the agar parallel to the line of separated proteins. Monoclonal or polyclonal antisera are loaded into the trough, and the gels are incubated to allow the antigens and antibodies to diffuse toward each other. Precipitation lines become visible when a zone of equivalence is reached. Typically, control serum is run above the trough, and the patient sample is run below the trough for easy comparison (Web Color Image 5–16).
2. Immunofixation electrophoresis (IFE). After serum, urine, or CSF samples are electrophoresed in an agarose gel, cellulose acetate impregnated with antiserum is placed on the gel. The antiserum from the cellulose acetate diffuses into the gel, and antigen-antibody precipitates form. The cellulose acetate is stained to visualize the precipitation bands. IFE and IEP are often used together to work up monoclonal gam- mopathies.
D. Labeling immunoassays can be qualitative or quantitative and can occur in the soluble or solid phase. Radioactive, enzyme, chemiluminescent, or fluorescent labels can be used.
1. RIA is a rapid and sensitive method that can be used to detect small amounts of antigen or antibody. However, exposure to radioisotopes can damage DNA and lead to radiation sickness, an increased incidence of neoplasms, or death. These potential hazards are a disadvantage to the RIA method (Web Color Image 5–17).
2. Radioallergosorbent test (RAST) is an RIA method specifically designed to measure antigen-specific IgE.
3. Radioimmunosorbent test (RIST) is a competitive binding technique used to quan- titate IgE.
4. ELISA is similar in principle to RIA and has the same sensitivity, but uses an enzyme label instead of a radioactive label. HRP and alkaline phosphatase (ALP) are the most commonly used enzymes. ELISAs can be used to detect extremely small amounts of antigen or antibody (Web Color Image 5–18).
5. IFA are often used to identify antigens in tissue sections or air-dried smears of pe- ripheral blood, bone marrow aspirates, touch preparations, or fine needle aspirate sam- ples.
a. Labeling. Fluorescein isothiocyanate is the most commonly used fluorescent la- bel. Labeling may be direct (using a labeled antibody) or indirect (using a labeled secondary antibody). After staining, the slides are washed and dried and immedi- ately examined using a fluorescence microscope. Fluorescence quenches quickly when exposed to light, which is the major disadvantage of immunofluorescence methods (Web Color Image 5–19).
b. Immunocytochemical techniques are modifications of the direct and indirect im- munofluorescence methods; enzyme labels such as HRP and ALP are used instead of fluorescent labels. These reaction products do not fade, so slides may be stored to provide permanent records.
E. Nephelometry (turbidimetry) photometrically measures the turbidity of solutions created by particles in suspension. It is accurate, rapid, and precise and is often used to quanti- tate immunoglobulins, complement components, and immune complexes. This method can also be used to measure antigen concentration. The light source in nephelometry instruments produces a wave-length of 840 nm.
F. Neutralization tests. Neutralizing antibodies can destroy the infectivity of viruses, which provides the basis for assays that can determine the amount of viral antibody present. These techniques are often used to detect antibodies against herpes simplex virus Types 1 and 2 (HSV-1 and HSV-2) and echovirus.
G. Cellular assays. A wide variety of techniques exists for assessing the function of the cells of the immune system.
1. Functional assays. These tests are rarely performed except in large medical centers and reference laboratories. Some tests are even considered obsolete. Functional assays include the following.
a. ADCC assays use bacteria-infected tumor cells to assess the killing ability of NK cells.
b. The Boyden chamber uses chemotactic substances to assess the chemotactic re- sponse of neutrophils.
c. Cell-mediated monocytolysis uses tumor cells to assess the killing functions of monocytes.
d. Latex bead ingestion assesses phagocytic activity.
e. Lymphocyte transformation tests assess the ability of lymphocytes to response to mitogens or specific antigens.
f. Lympholysis tests assess the ability of Tcto lyse labeled target cells.
g. Microcytotoxicity studies are used to detect HLA antigens and antibodies.
h. Migration inhibition techniques determine the ability of lymphocytes to produce chemotactic factors in response to granulocytes and monocytes.
i. Mixed-lymphocyte cultures are used to assess the human leukocyte antigen D (HLA-D) compatibility of donor and recipient lymphocytes.
j. NBT reductase tests are used to assess the intracellular killing ability of neu- trophils.
k. Phagocytosis assays mix bacteria with neutrophils to assess the cells’ phagocytic ability.
2. Flow cytometry can be used to identify subpopulations of cells such as reticulocytes, granulocytes, T-cell subsets, B lymphocytes, and others. Fluorescent dyes, such as fluorescein, acridine orange, and phycoerythrin, are bound specifically to the cell marker of interest. Cells in suspension pass singly through a laminar-flow saline sheath.
As the stained cells pass through a laser beam (usually argon or krypton), the dye is activated, and the cell fluoresces. The fluorescence is detected and collected by sensors placed at 90◦relative to the source beam, and the information is processed by a computer (Web Color Image 5–20).