Immunological Testing

The antigen and antibody reaction has been used for decades for detecting and characterizing microorganisms and their components in medical, food, and diagnostic microbiology. This reaction is the basis for serotyping bacteria such as salmonella, Escherichia coli O157:H7, and Listeria monocytogenes. These antibodies can be polyclonal (a mixture of several antibodies in the antisera which can react with different sites of the antigens) or monoclonal (only one pure antibody in the antiserum which will react with only one epitope of the antigens). Both polyclonal and monoclonal antibodies have been used extensively in applied food microbiology. There are many ways to perform antigen-antibody reactions, but the most popular format in recent years has been the "sandwich" enzyme-linked immunosorbant assay, popularly known as the ELISA test.

Briefly, antibodies (e.g., anti-salmonella antibody) are fixed on a solid support (e.g., wells of a microtiter plate). A solution containing a suspect target antigen (e.g., salmonella) is introduced to the microtiter well. If the solution has salmonella antigens, it will be captured by the immobilized antibodies.

After washing away food debris and excess materials, another anti-salmonella antibody complex is added into the solution. The second anti-salmonella antibody will react with another part of the trapped salmonella. This second antibody is linked with an enzyme such as horseradish peroxi-dase. After another washing to remove debris, a chromagen complex such as tetramethylbenzidine and hydrogen peroxide is added. The enzyme will react with the chromagen and will produce a colored compound that will indicate that the first antibody has captured salmonella. If all the reaction procedures are done properly and the liquid in a microtiter well exhibits a color reaction, then the sample is considered positive for salmonella.

This procedure is simple to operate and has been used for decades with excellent results. It should be emphasized that these ELISA tests need about a million cells to be reactive, and therefore, before performing the ELISA tests, the food sample has to go through an overnight incubation so that the target organism reaches a detectable level. The total time to detect pathogens by these systems includes the enrichment time of the target pathogens (ca. 24 hrs).

Many diagnostic companies (such as BioControl, Organon Teknika, and Tecra) have marketed ELISA test kits for foodborne pathogens and toxins (e.g., salmonella, Escherichia coli) and toxins (e.g., staphylococcal enterotox-ins). However, the time involved in sample addition, incubating, washing and discarding of liquids, adding of another antibody complex, washing, and, finally, adding of reagents for color reaction all contribute to the inconvenience of the manual operation of the ELISA test. Recently several companies have completely automated the entire ELISA procedure.

VIDAS (bioMerieux, Hazelwood, MO) is an automated system which can perform the entire ELISA procedure automatically and can complete an assay in 45 minutes to 2 hours, depending on the test kit. Since VIDAS utilizes a more sensitive fluorescent immunoassay for reporting the results, the system is named enzyme-linked fluorescent assay (ELFA). All the analyst needs to do is to present to the reagent strip a liquid sample of an overnight enriched sample. The reagent strip contains all the necessary reagents in a ready-to-use format. The instrument will automatically transfer the sample into a plastic tube called the solid phase receptacle (SPR) which contains antibodies to capture the target pathogen or toxin. The SPR will be automatically transferred to a series of wells in succession to perform the ELFA test. After the final reaction, the result can be read, and interpretation of a positive or negative test will be automatically determined by the instrument. Presently, VIDAS can detect listeria, Listeria monocytogenes, salmonella, E. coli O157, staphylococcal enterotoxin, and campylobacter. Its manufacturers also market an immuno-concentration kit for salmonella and E. coli O157. Currently more than 13,000 VIDAS units are in use internationally.

BioControl (Bellevue, WA) markets an enzyme immunoassay (EIA) system called Assurance EIA which can be adapted to automation for high-volume testing. Assurance EIA is available for salmonella, listeria, E. coli O157:H7, and campylobacter. Diffchamb (Hisings Backa, Sweden) has a high-precision liquid delivery system that can be used to perform a variety of ELISA tests depending on the pathogens to be tested. Tecra OPUS (International BioProducts, Redmond, WA) and Bio-Tek (Highland Park, VT) instruments can also perform ELISA tests automatically as long as the proper reagents are applied to the system. Many ELISA test kits are now highly standardized and the test can be performed automatically to increase efficiency and reduce human errors.

Another exciting development in immunology is the use of lateral flow technology to perform antigen-antibody tests. In this system, the unit has three reaction regions. The first well contains antibodies to react with target antigens. These antibodies have color particles attached to them. A liquid sample (after overnight enrichment) is added to this well, and if the target organism (e.g. E. coli O157:H7) is present, it will react with the antibodies. The complex will migrate laterally by capillary action to the second region which contains a second antibody designed to capture the target organism. If the target organism is present, the complex will be captured, and a blue line will form due to the color particles attached to the first antibody. Excess antibodies will continue to migrate to the third region which contains another antibody that reacts with the first antibody (which has now become an antigen) and will form a blue color band. This is a "control" band indicating that the system is functioning properly. The entire procedure takes only about 10 minutes. This is truly a rapid test!

Neogen (Lansing, MI; Reveal system) and BioControl (Bellevue, WA; VIP system) are the two main companies marketing this type of system for E. coli O157, salmonella, and listeria. Merck KGaA (Darmstadt, Germany) developed similar systems using gold particles in the reagent to increase the sensitivity of the test.

A number of interesting methods utilizing growth of the target pathogen are also available to detect antigen-antibody reactions. The BioControl 1-2 test (BioControl, Bellevue, WA) is designed to detect motile salmonella from foods. In this system, the food sample is first preenriched for 24 hours in a broth, and then 0.1 ml is inoculated into one of the chambers in an L-shaped system. The chamber contains selective enrichment liquid medium for salmonella. There is a small hole connecting the liquid chamber with a soft agar chamber through which salmonella can migrate. An opening on the top of the soft agar chamber allows the analyst to deposit a drop of polyvalent anti-H antibodies against flagella of salmonella. The antibodies move downward in the soft agar due to gravity and diffusion. If salmonella is present, it will migrate throughout the soft agar. As the salmonella and the anti-H antibodies meet, they will react and form a visible V-shaped "immunoband." The presence of the immunoband indicates the presumptive positive for salmonella in the food sample. This reaction occurs after overnight incubation of the unit. This system is easy to use and interpret, and it has gained popularity because of its simplicity.

Tecra (Roseville, Australia) developed a detection system (Unique Salmonella) that combines immuno-capturing, growth of the target pathogen, and an ELISA test in a simple-to-use self-contained unit. The food is first preenriched in a liquid medium overnight and an aliquot is added into the first tube of the unit. Into this tube a dipstick coated with salmonella antibodies is introduced and left in place for 20 minutes; at this time the antibodies will capture salmonella, if present. The dipstick, with salmonella attached, is then washed and placed into a tube containing growth medium. The dipstick is left in this tube for 4 hours. During this time, if salmonella is present, it replicates, and the newly produced salmonella are automatically trapped by the coated antibodies. Thus, after 4 hours of replication, the dipstick becomes saturated with trapped salmonella. The dipstick is then transferred to another tube containing a second antibody conjugated to enzyme, and the tube contents are allowed to react for 20 minutes. After this second antigen-antibody reaction, the dipstick is washed in the fifth tube and placed into the last tube for color development similar to other ELISA tests.

Development of a purple color on the dipstick indicates the presence of salmonella in the food. The entire process, from incubation of food sample to reading of the test results, requires about 22 hours, making it an attractive system for detection of salmonella. A similar system can now also detect listeria. An automated system is now being marketed.

The BioControl 1-2 test and the Unique Salmonella test are designed for laboratories with a low volume of tests. Thus, both the automatic systems and the hands-on unit systems have their place in different food testing laboratory situations.

A truly innovative development in applied microbiology is the immuno-magnetic separation system. Vicam (Somerville, MA) pioneered this concept by coating antibodies against listeria on metallic particles. Large numbers of these particles (in the millions) are added into a liquid suspected to contain listeria cells. The antibodies on the particles will capture the listeria cells while the mixture is rotated for about an hour. After the reaction has gone to completion, the tube is placed next to a powerful magnet which will immobilize all the metallic particles at the side of the glass test tube regardless of whether the particles have or have not captured the listeria cells. The rest of the liquid will be decanted. By removing the magnet from the tube, the metallic particles can again be suspended in a liquid. At this point, the only cells in the solution will be the captured listeria. By introducing a smaller volume of liquid (e.g., 10% of the original volume), the cells are now concentrated by a factor of 10. Cells from this liquid can be detected by direct plating on selective agar, ELISA tests, PCR reaction, or other microbiological procedures in almost pure culture state. Immunomagnetic capture can save at least one day in the total protocol of preenrichment and enrichment steps of pathogen detection in food.

Dynal (Oslo, Norway) developed this concept further by use of very homogeneous paramagnetic beads that can carry a variety of molecules such as antibodies, antigens, and DNA. Dynal has developed beads to capture E. coli O157, listeria, cryptosporidium, giardia, and others. Furthermore, the beads can be supplied without any coating materials, and scientists can tailor them to their own needs by coating with the necessary antibodies or other capturing molecules for detection of target organisms. Currently, many diagnostic systems (ELISA, PCR, etc.) are incorporating an immunomagnetic capture step to reduce incubation and increase sensitivity of the entire protocol.

Fluorescent antibody techniques have been used for decades for the detection of salmonella and other pathogens. Similar to the DEFT test designed for viable cell count, fluorescent antibodies can be used to detect a great variety of target microorganisms such as E. coli O157:H7 in milk and juice.

One of the newest and fastest immunological methods to detect food-borne pathogens is the Pathatrix system (Matrix MicroScience, Golden, CO). Wu et al. [16] tested a same-day protocol for the detection of Escherichia coli O157:H7 by the Pathatrix system (which employs a novel immuno-capture method) and Colortrix (a rapid ELISA test). The Pathatrix system can circulate a 4.5 hour preenriched 250 ml sample (25 g of food in 225 ml of preenrichment broth) over a sheet of paramagnetic beads coated with antibodies against E. coli O157:H7 many times in 30 minutes to capture almost all target pathogens. This circulation system increased the concentration of E. coli O157:H7 from the population after 4.5 hours of enrichment to 1.2 to 2.6log CFU/25g higher concentration in 30 minutes. After Pathatrix concentration the beads with target pathogens are applied to the Colortrix system, a rapid ELISA system that was able to detect E. coli O157:H7 in 15 minutes. The results indicated an excellent correlation (100%) between positive Pathatrix/Colortrix (5.25 hours) compared with a 30-hour conventional plating method. The sensitivity of the system is from 0.7 to 2.1 log CFU/25 g as the initial concentration of E. coli O157:H7 in the sample before the 4.5 hours of enrichment. This system is also able to detect Listeria monocytogenes, campylobacter, and other pathogens.

Antigen-antibody reaction provides a powerful system for rapid detection of all kinds of pathogens and molecules. This section has described some of the useful methods developed for applied food microbiology. Some systems are highly automated, and others are exceedingly simple to operate. It should be emphasized that many of the immunological tests described in this section provide presumptive positive or presumptive negative screening test results. For negative screening results, the food in question is allowed to be shipped for commerce. For presumptive positive test results, the food will not be allowed for shipping until confirmation of the positive is done by the conventional microbiological methods.

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