20.1 Polyclonal and Monoclonal Antibody Production
- Antibodies bind with high specificity to antigens used to challenge the immune system, but they may also show cross-reactivity by binding to other antigens that share chemical properties with the original antigen.
- Injection of an antigen into an animal will result in a polyclonal antibody response in which different antibodies are produced that react with the various epitopes on the antigen.
- Polyclonal antisera are useful for some types of laboratory assays, but other assays require more specificity. Diagnostic tests that use polyclonal antisera are typically only used for screening because of the possibility of false-positive and false-negative results.
- Monoclonal antibodies provide higher specificity than polyclonal antisera because they bind to a single epitope and usually have high affinity.
- Monoclonal antibodies are typically produced by culturing antibody-secreting hybridomas derived from mice. mAbs are currently used to treat cancer, but their exorbitant cost has prevented them from being used more widely to treat infectious diseases. Still, their potential for laboratory and clinical use is driving the development of new, cost-effective solutions such as plantibodies.
20.2 Detecting Antigen-Antibody Complexes
- When present in the correct ratio, antibody and antigen will form a precipitin, or lattice that precipitates out of solution.
- A precipitin ring test can be used to visualize lattice formation in solution. The Ouchterlony assay demonstrates lattice formation in a gel. The radial immunodiffusion assay is used to quantify antigen by measuring the size of a precipitation zone in a gel infused with antibodies.
- Insoluble antigens in suspension will form flocculants when bound by antibodies. This is the basis of the VDRL test for syphilis in which anti-treponemal antibodies bind to cardiolipin in suspension.
- Viral infections can be detected by quantifying virus-neutralizing antibodies in a patient’s serum.
- Different antibody classes in plasma or serum are identified by using immunoelectrophoresis.
- The presence of specific antigens (e.g., bacterial or viral proteins) in serum can be demonstrated by western blot assays, in which the proteins are transferred to a nitrocellulose membrane and identified using labeled antibodies.
- In the complement fixation test, complement is used to detect antibodies against various pathogens.
20.3 Agglutination Assays
- Antibodies can agglutinate cells or large particles into a visible matrix. Agglutination tests are often done on cards or in microtiter plates that allow multiple reactions to take place side by side using small volumes of reagents.
- Using antisera against certain proteins allows identification of serovars within species of bacteria.
- Detecting antibodies against a pathogen can be a powerful tool for diagnosing disease, but there is a period of time before patients go through seroconversion and the level of antibodies becomes detectable.
- Agglutination of latex beads in indirect agglutination assays can be used to detect the presence of specific antigens or specific antibodies in patient serum.
- The presence of some antibacterial and antiviral antibodies can be confirmed by the use of the direct Coombs’ test, which uses Coombs’ reagent to cross-link antibodies bound to red blood cells and facilitate hemagglutination.
- Some viruses and bacteria will bind and agglutinate red blood cells; this interaction is the basis of the direct hemagglutination assay, most often used to determine the titer of virus in solution.
- Neutralization assays quantify the level of virus-specific antibody by measuring the decrease in hemagglutination observed after mixing patient serum with a standardized amount of virus.
- Hemagglutination assays are also used to screen and cross-match donor and recipient blood to ensure that the transfusion recipient does not have antibodies to antigens in the donated blood.
20.4 EIAs and ELISAs
- Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen.
- Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular structures (immunocytochemistry).
- Direct ELISA is used to quantify an antigen in solution. The primary antibody captures the antigen, and the secondary antibody delivers an enzyme. Production of end product from the chromogenic substrate is directly proportional to the amount of captured antigen.
- Indirect ELISA is used to detect antibodies in patient serum by attaching antigen to the well of a microtiter plate, allowing the patient (primary) antibody to bind the antigen and an enzyme-conjugated secondary antibody to detect the primary antibody.
- Immunofiltration and immunochromatographic assays are used in lateral flow tests, which can be used to diagnose pregnancy and various diseases by detecting color-labeled antigen-antibody complexes in urine or other fluid samples
20.5 Fluorescent Antibody Techniques
- Immunofluorescence assays use antibody-fluorogen conjugates to illuminate antigens for easy, rapid detection.
- Direct immunofluorescence can be used to detect the presence of bacteria in clinical samples such as sputum.
- Indirect immunofluorescence detects the presence of antigen-specific antibodies in patient sera. The fluorescent antibody binds to the antigen-specific antibody rather than the antigen.
- The use of indirect immunofluorescence assays to detect antinuclear antibodies is an important tool in the diagnosis of several autoimmune diseases.
- Flow cytometry uses fluorescent mAbs against cell-membrane proteins to quantify specific subsets of cells in complex mixtures.
- Fluorescence-activated cell sorters are an extension of flow cytometry in which fluorescence intensity is used to physically separate cells into high and low fluorescence populations.