Immunoassays

Immunoassays are based on the principles that specific antigens will stimulate very specific and unique immune responses and that the proteins produced by the immune response, called antibodies, can be used to signal the presence of a target compound in a sample. The principle of an indirect EIA for antibody is when an antibody in the specimen is bound by a "capture antigen" which has been attached to a solid surface. The bound antibody is detected by binding an added enzyme conjugated anti- human antibody. The bound enzyme labeled antibody is detected by its ability to break down its substrate to a coloured product. Although enzyme immunoassays can be set up in several different formats the indirect method is the most appropriate for the detection of total specific antibody.

TLC

Chromatography is a method of separating mixtures of two or more compounds. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned.

Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). Solids most commonly used in chromatography are silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O). Both of these adsorbents are polar, but alumina is more so. Silica is also acidic. Alumina is available in neutral, basic, or acidic forms.

TLC involves spotting the sample to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent the stationary adsorbent phase. The more strongly a given component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the plate.

Rapid Methods

The rapid detection of pathogens and other microbial contaminants in food is critical for ensuring the safety of consumers. Traditional methods to detect foodborne bacteria often rely on time-consuming growth in culture media, followed by isolation, biochemical identification, and sometimes serology. Recent advances in technology make detection and identification faster, more convenient, more sensitive, and more specific than conventional assays -- at least in theory. These new methods are often referred to as "rapid methods", a subjective term used loosely to describe a vast array of tests that includes miniaturized biochemical kits, antibody- and DNA-based tests, and assays that are modifications of conventional tests to speed up analysis. Some of these assays have also been automated to reduce hands-on manipulations. With few exceptions, almost all assays used to detect specific pathogens in foods require some growth in an enrichment medium before analysis.

There are many DNA-based assay formats, but only probes, PCR and bacteriophage have been developed commercially for detecting foodborne pathogens. Probe assays generally target ribosomal RNA (rRNA), taking advantage of the fact that the higher copy number of bacterial rRNA provides a naturally amplified target and affords greater assay sensitivity.

The basic principle of DNA hybridization is also being utilized in other technologies, such as the polymerase chain reaction (PCR) assay, where short fragments of DNA (probes) or primers are hybridized to a specific sequence or template, which is then enzymatically amplified by Taq polymerase using a thermocycler. Theoretically, PCR can amplify a single copy of DNA by a million fold in less than 2 hrs; hence its potential to eliminate, or greatly reduce the need for cultural enrichment. However, the presence of inhibitors in foods and in many culture media can prevent primer binding and diminish amplification efficiency, so that the extreme sensitivity achievable by PCR with pure cultures is often reduced when testing foods. Therefore, some cultural enrichment is still required prior to analysis.

The enzyme-linked immunosorbent assay (ELISA) is the most prevalent antibody assay format used for pathogen detection in foods. Usually designed as a "sandwich" assay, an antibody bound to a solid matrix is used to capture the antigen from enrichment cultures and a second antibody conjugated to an enzyme is used for detection. The walls of wells in microtiter plates are the most commonly used solid support; but ELISAs have also been designed using dipsticks, paddles, membranes, pipet tips or other solid matrices.

Applications and Limitations of Rapid Methods
Almost all rapid methods are designed to detect a single target, which makes them ideal for use in quality control programs to quickly screen large numbers of food samples for the presence of a particular pathogen or toxin. A positive result by a rapid method however, is only regarded as presumptive and must be confirmed by standard methods. Although confirmation may extend analysis by several days, this may not be an imposing limitation, as negative results are most often encountered in food analysis. Most rapid methods can be done in a few minutes to a few hours, so they are more rapid than traditional methods. But, in food analysis, rapid methods still lack sufficient sensitivity and specificity for direct testing; hence, foods still need to be culture-enriched before analysis. Although enrichment is a limitation in terms of assay speed, it provides essential benefits, such as diluting the effects of inhibitors, allowing the differentiation of viable from non-viable cells and allowing for repair of cell stress or injury that may have resulted during food processing.

Electroimmunoessays

Electroimmunoassays consists of a circuit with a capture antibody which is attached to the solid surface of the electrode gap area. This technology to isolate and identify foodborne pathogens combines specific antibody-antigen binding to the production of an electrical signal. The target antigen binds to the capture antibody when the sample is added. Following that, a colloidal gold–labeled detection antibody is bound to create a capture-target-detector sandwich. Finally, the silver ions are deposited onto the colloidal gold, which produces a conductive silver bridge hence closing the circuit and resulting in a measured drop in resistance.

DNA Probe

The identification of bacteria by DNA probe hybridization methods is based on the presence or absence of particular genes. This is in contrast to most biochemical and immunological tests that are based on the detection of gene products such as antigens or chemical end products of a metabolic pathway. A DNA probe is composed of nucleic acid molecules, most often double-stranded DNA. It consists of either an entire gene or a fragment of a gene with a known function. Alternatively, short pieces of single-stranded DNA can be synthesized, based on the nucleotide sequence of the known gene. These are commonly referred to as oligonucleotides. Both natural and synthetic oligonucleotides are used to detect complementary DNA or RNA targets in samples. Double-stranded DNA probes must be denatured before the hybridization reaction; oligonucleotide and RNA probes, which are single-stranded, do not need to be denatured. Target nucleic acids are denatured by high temperature or high pH, and then the labeled gene probe is added. If the target nucleic acid in the sample contains the same nucleotide sequence as that of the gene probe, the probe will form hydrogen bonds with the target. Thus the labeled probe becomes specifically associated with the target. The unreacted, labeled probe is removed by washing the solid support, and the presence of probe-target complexes is signaled by the bound label.

The physical basis for gene probe tests stems from the structure of DNA molecules themselves. Usually, DNA is composed of two strands of nucleotide polymers wound around each other to form a double helix. These long nucleotide chains are held together by hydrogen bonds between specific pairs of nucleotides. The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. Single-stranded molecules result and the DNA is considered denatured. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.

Laboratory Methods

DNA and Protein-based methods have been developed and applied for the detection of GMO. For the detection of Genetically Modified Organisms (GMOs) at the level of DNA, PCR based methods are mainly used, whereas for protein-based detection, immunoassays such as Western blot, ELISA, and lateral strips methods are predominantly used.

DNA -based methods are primarily based on multiplying a specific DNA with the PCR technique. The PCR reaction allows the million or billion fold amplification of a specific target DNA fragment framed by two synthetic oligonucleotide primers. The PCR is done by a multiple-process with consecutive cycles of denaturation step to denature DNA, annealing step where primers find their correspondent complementary sequences on the template and extension or elongation step where Taq polymerase copies the complementary strand. In each cycle three different steps were progressed in different temperatures. To perform the reliable PCR test, it is important that specific primers were designed to bind selectively to the complementary sequences of the target DNA; the specific gene introduced, promoter and terminator, a sequence spanning between a regulatory sequence and a sequence between the gene and the flanking genome A prerequisite for the PCR-based technique is to know specific DNA sequence inserted in GMO, served as target and to find adequate standard materials to be used as positive analytical controls. Three kinds of PCR strategies are currently used for GMO detection; multiplex PCR, quantitative competitive PCR (QC-PCR) and real-time PCR.

Immunoassay is based on the specific binding between an antigen and an antibody. Thus, the availability of antibodies with the desired affinity and specificity is the most important factor for setting up immunoassay systems. This technology is ideal for qualitative and quantitative detection of many types of proteins, however can’t be applied in highly processed soy products

ELISA is an enzyme linked immuosorbant assay. This technique uses an antibody that recognizes specific proteins. Protein is bound to a well of a plastic plate instead of a membrane. Antigen and antibody bind and produce a stable complex, which can be visualized by addition of a second antibody linked to an enzyme. The advantages of ELISA are to be capable of quantitative analysis when a standard curve is included, and high throughput.