Antibody-Based Identification Methods
Antibody-based identification methods have been used in some form for decades. They are now commonly used for the rapid detection and identification of microbes in food microbiology. Antigens are protein compounds that bind to particular cell structures or derivatives that are usually harmful. Antibodies are made to recognize these harmful structures and tag them for degradation in the body. The antibody-antigen reaction can be very specific and therefore can be used to quantitate the level of antigen.
Immunoflorescence was one of the first methods used in rapid identification. Antibodies are linked with fluorescent dyes and can be combined, then fixed, with microbes. After the unbound reagent is washed off, the slide can be viewed under microscope with fluorescent light and particular antigenic cells or structures can be easily distinguished. Latex agglutination combines a similar method coupling antibodies to latex microspheres. This antibody latex combination creates a complex with microbial cells and is visible as a clump. The issue with this method is that its sensitivity is in the range of 107-108 cells. Greater sensitivity is necessary in many applications. Reverse passive latex agglutination (RPLA) employs similar methods to the previous one but observing the pattern created in a tube after the complex is formed (Downes and Ito, 2001).
The most common and now standard of identification techniques for microorganisms and their products is an enzyme immunoassay (EIA) or enzyme-linked immunoabsorbent assay (ELISA). Microbes can be characterized by their genetic composition through a number of methods. First, a “fingerprint” of the organism must be procured from isolated genetic material. Once pure colonies have been isolated from a culture in medium, they are inactivated by heat. Through the use of a sample carrier, DNA can be extracted from the cell lysate and then digested to completion using a restriction enzyme, EcoR1. Using electrophoresis, the DNA fragments are differentiated by their molecular size and can be transferred to moving nylon membranes or another solid phase in a “sandwich”. A chemically labeled rRNA operon probe derived from E. coli is hybridized with the sample after they are denatured. Then, the membrane is washed with a combination of an antibody alkaline phosphatase conjugate and blocking buffer. The unbound antibody conjugate can be removed, allowing for the attachment of either an electro- or chemiluminescent substrate. Wherever the conjugate was unbound, an intense pattern of luminescent DNA fragments can be compared. This pattern can then be compared to known patterns within a database (MI Labs).
Immunoprecipitate detection is a new entry into the realm of rapid assays. A sample of the enriched culture is pushed through a “paddle” and across a chromatographic matrix. The matrix contains antibodies combined to a material that can precipitate, like latex or gold. The culture is combined with the antibody conjugate and is adsorbed across the matrix where it binds with a secondary, stationary antibody. The antigen-antibody-matrix precipitate falls out and is visible as a colored band across the matrix. In total, the process takes a minimal amount of time at five minutes (Downes and Ito, 2001).
Antibodies are used for their ability to separate target antigens in addition to the previously described ability of identification. Both immunoaffinity chromatography and immunomagnetic separation (IMS) accomplish this goal. The former can isolate certain particulates by coupling antibodies with a solid inert matrix, usually in a column, and allowing the culture to pass through, separating the antigen from other materials through a series of buffers. IMS reacts microbial cells with antibody-coated magnetic beads with their specific binding capacity. A magnet can then be applied to the side of a test tube and the target cells are separated from the rest of the medium (Downes and Ito, 2001).
Downes, Frances and Keith Ito. Compendium of Methods for the Microbiological Examination of Foods. Washington: American Public Health Association, 2001.