Genetically Modified foods
Genetically Modified foods are produced from genetically modified organisms (GMO) with their genome altered via genetic engineering techniques. The general principle to produce a GMO is to insert DNA from another organism (and modified in the laboratory) into an organism's genome. This produces both new and useful traits. Typically this is done using DNA from certain types of bacteria.

GM Foods, Detection

Benefits of Genetically Modified Foods:

1. Agricultural – increased yield
2. Environmental – reduced use of pesticides, herbicides and fuel
3. Nutritional – improved quality of food
4. Disease prevention – edible vaccines

Risks of Genetically Modified Foods:

1. Exposure to possible allergens and toxins
2. Harm to the environment
3. Antibiotic resistance
4. Spread of introduced genes to non-target plants by out crossing and pollen drift

Detection of Genetically Modified Foods

There are 2 basic means for detecting genetic modification.

1. Test food for the product of the transgene, usually a protein.

2. Test for the presence of DNA from the transgene or another portion of the gene cassette.

Enzyme Linked ImmunoSorbent Assay (ELISA) is an assayed protein. Enzyme linked to the antibody (bound to protein) will react with a coloured substrate, enabling detection of a specific protein.

Benefits of ELISA

• ELISA is cheaper then DNA tests
• Give rapid, faster results
• Can sometimes be done on the spot

Disadvantages of ELISA

• Does not work well on processed food (heat during processing may have destroyed the protein)
• DNA tests are very accurate, workable on processed food and can be quantified

ELISA (cont'd)

Introduction to Antibodies - Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA Procedure

Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number. ELISAs can provide a useful measurement of antigen or antibody concentration.

Sandwich ELISA Assays
One of the most useful of the immunoassays is the two antibody “sandwich” ELISA. This assay is used to determine the antigen concentration in unknown samples. This ELISA is fast and accurate, and if a purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample. The sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies.

To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate. Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding.

Unlike Western blots, which use precipitating substrates, ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3’5,5’- tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively. A table of commonly used enzyme-substrate combinations is included in Appendix D.

The Sensitivity of the Sandwich ELISA is Dependent on Four Factors:

1. The number of molecules of the first antibody that are bound to the solid phase.
2. The avidity of the first antibody for the antigen.
3. The avidity of the second antibody for the antigen.
4. The specific activity of the second antibody.

The amount of the capture antibody that is bound to the solid phase can be adjusted easily by dilution or concentration of the antibody solution. The avidity of the antibodies for the antigen can only be altered by substitution with other antibodies. The specific activity of the second antibody is determined by the number and type of labeled moieties it contains.

General Protocol for the Sandwich ELISA method:

1. Before the assay, both antibody preparations should be purified and one must be labeled.
   
2. For most applications, a polyvinylchloride (PVC) microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.
   
3. Bind the unlabeled antibody to the bottom of each well by adding approximately 50 µL of antibody solution to each well (20 µg/mL in PBS). PVC will bind approximately 100ng/well (300 ng/cm²). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 µg/well. This is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again.
   
4. Incubate the plate overnight at 4°C to allow complete binding.
   
5. Wash the wells twice with PBS. A 500 mL squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.
   
6. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs. to overnight in a humid atmosphere at room temperature.
   
   Note: Sodium azide is an inhibitor or horseradish peroxidase. Do not include sodium azide in buffers or wash solutions if an HRP-labeled antibody will be used for detection.
   
7. Wash wells twice with PBS.
   
8. Add 50 µL of the antigen solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS). Incubate for at least 2 hrs. at room temperature in a humid atmosphere.
   
9. Wash the plate four times with PBS.
   
10. Add the labeled second antibody. The amount to be added can be determined in preliminary experiments. For accurate quantitation, the second antibody should be used in excess. All dilutions should be done in the blocking buffer.
    
11. Incubate for 2 hrs. or more at room temperature in a humid atmosphere.
    
12. Wash with several changes of PBS.
    
13. Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA plate reader.

Note: Some enzyme substrates are considered hazardous, due to potential carcinogenicity. Handle with care and refer to Material Safety Data Sheets for proper handling precautions.

For quantitative results, compare signal of unknown samples against those of a standard curve. Standards must be run with each assay to ensure accuracy.

Competitive ELISA Assays
When two “matched pair” antibodies are not available for your target, another option is the competitive ELISA. Another advantage to the competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISAs, below is an example for one such configuration. In order to utilize a competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the immunogen or the primary antibody. The protocol below uses a labeled immunogen as the competitor. For other configurations of competitive ELISAs, see Appendix F, Harlow and Lane (1996).

Briefly, an unlabeled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards and unknowns. After this reaction is allowed to go to equilibrium, conjugated immunogen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled immunogen. Thus, the more immunogen in the sample or standard, the lower the amount of conjugated immunogen bound. The plate is then developed with substrate and color change is measured.

General Protocol for the Competitive ELISA Method:

1. For most applications, a polyvinylchloride (PVC) microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.
   
2. Add 50 µL of diluted primary antibody (capture) to each well. The appropriate dilution should be determined using a checkerboard titration prior to testing samples. PVC will bind approximately 100 ng/well (300 ng/cm²). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 µg/well. this is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again. Allow to incubate for 4 hrs. at room temperature or 4°C overnight.
   
   Note: If a purified capture antibody is not available, the plate should first be coated with a purified secondary antibody directed against the host of the capture antibody according to the following procedure:
   
   A. Bind the unlabeled secondary antibody to the bottom of each well by adding approximately 50 µL of antibody solution to each well (20 µg/mL in PBS).
   B. Incubate the plate overnight at 4°C to allow complete binding.
   C. Add primary capture antibody (as above).
   
3. Wash the wells twice with PBS. A 500 mL squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.
   
4. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs. to overnight in a humid atmosphere at room temperature.
   
5. Wash wells twice with PBS.
   
6. Add 50 µL of the standards or sample solution to the wells. All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20).
   
   Note: Sodium azide is an inhibitor or horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection.
   
7. Add 50 µL of the antigen-conjugate solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20). Incubate for at least 2 hrs. at room temperature in a humid atmosphere.
   
8. Wash the plate four times with PBS.
   
9. Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.
   

Note: Competitive ELISAs yield an inverse curve, where higher values of antigen in the samples or standards yield a lower amount of color change.

Troubleshooting ELISA Assays:

• Interpret the control results.
  
• If the negative controls are giving positive results, there may be contamination of the substrate solution, or contamination of the enzyme-labeled antibody, or of the controls themselves.
  
• If no color has developed for the positive controls or for the samples, check all reagents for dating, concentration, and storage conditions. Check the integrity of the antibody reagent.
  
• If very little color has developed for the positive controls and the test samples, check the dilution of the enzyme labeled antibody, and the concentration of the substrate.
  
• If color has developed for the test samples but not the positive or negative controls, check the source of the positive controls, their expiration date and their storage. Have they been stored in a dilute form, so that the antigen may have adhered to the surface of the storage vessel?
  
• If color can be seen, but the absorbance is not as high as expected, check the wavelength setting.
  
• When rerunning an assay while troubleshooting, change only one factor at a time.

ELISA

Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with mild detergent to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

Polymerase Chain Reaction (PCR)

PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

Procedure
The PCR usually consists of a series of 20 to 35 cycles. Most commonly, PCR is carried out in three steps (Fig. 2), often preceded by one temperature hold at the start and followed by one hold at the end.

1. Prior to the first cycle, during an initialization step, the PCR reaction is often heated to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), and this temperature is then held for 1-9 minutes. This first hold is employed to ensure that most of the DNA template and primers are denatured, i.e., that the DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding two single strands of DNA. Also, some PCR polymerases require this step for activation. Following this hold, cycling begins, with one step at 94-98°C for 20-30 seconds (denaturation step).
2. The denaturation is followed by the annealing step. In this step the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template. Brownian motion causes the primers to move around, and DNA-DNA hydrogen bonds are constantly formed and broken between primer and template. Stable bonds are only formed when the primer sequence very closely matches the template sequence, and to this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis. The temperature at this step depends on the melting temperature of the primers, and is usually between 50-64°C for 20-40 seconds.
3. The annealing step is followed by an extension/elongation step during which the DNA polymerase synthesizes new DNA strands complementary to the DNA template strands. The temperature at this step depends on the DNA polymerase used. Taq polymerase has a temperature optimum of 70-74°C; thus, in most cases a temperature of 72°C is used. The hydrogen bonds between the extended primer and the DNA template are now strong enough to withstand forces breaking these attractions at the higher temperature. Primers that have annealed to DNA regions with mismatching bases dissociate from the template and are not extended. The DNA polymerase condenses the 5'- phosphate group of the dNTPs with the 3'- hydroxyl group at the end of the nascent (extending) DNA strand, i.e., the polymerase adds dNTP's that are complementary to the template in 5' to 3' direction, thus reading the template in 3' to 5' direction. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute. A final elongation step of 5-15 minutes (depending on the length of the DNA template) after the last cycle may be used to ensure that any remaining single-stranded DNA is fully extended. A final hold of 4-15°C for an indefinite time may be employed for short-term storage of the reaction, e.g., if reactions are run overnight.

(http://en.wikipedia.org/wiki/Polymerase_chain_reaction)

Crabsticks

Crab sticks are processed sea food made of surimi (or most of the time finely pulverized white fish flesh). It is shaped and cured to resemble snow crab legs. They are usually coloured red and rectangular-oblong in shape. Small strings of the crab sticks can be neatly pulled and torn out in a similar manner to string cheese. The smell of crab sticks is similar to seafood products, having a sweet, salty and highly umami taste. Crab sticks are cooked in its curing process and they can be eaten directly from a package.

Process

First, lean meat from fish is separated or minced. In order to eliminate undesirable odours, the meat may then be rinsed several times. The resulting meat is then beaten and pulverized to form a gelatinous paste. The gelatinous paste is mixed with differing proportions of additives such as starch, egg white, salt, sugar, vegetable oil, sorbitol, soy protein and seasonings, depending on the desired texture and flavour of the final product If the surimi is to be packed and frozen, food-grade cryoprotectants are added while the meat paste is being mixed. Under most circumstances, surimi is immediately processed, formed and cured into surimi products at the time it is produced.

Seafood Spoilage Causes

Spoilage causes

• Bacteria

Bacteria is the main cause of seafood spoilage. Millions of bacteria are present in the surface slime, gills, and gut of living seafood species. When seafood species die, they produce fishy odours and flavours, and discolourations. This is caused by bacteria. They invade the flesh through the gills, along blood vessels, and directly through the skin and belly cavity lining in the flesh. If food poisoning bacteria are present, they can multiply and cause illness when the seafood is eaten.

• Enzymes

Many different enzymes are present in living seafood species. They help build tissue, contract and relax muscles, and digest food. When seafood species die, enzymes continue to work and start to breakdown the flesh. This causes the flesh to soften and lowering the quality. Enzymes also produce more food for bacteria to feed on, increasing rate of spoilage.

• Chemical Action

Oxygen in the air can attack unsaturated oils or, seafood causing rancidity, off-odors and off-flavors. This is especially important in fatty fish such as salmon and mackerel.

• Slowing Seafood Spoilage

High temperatures speed spoilage and low temperatures slow spoilage. When temperature is increased from 32ºF to 40ºF, it will double the rate of spoilage and cuts the shelf life in half.

Sanitation is also important. Contamination of seafood by bacteria from dirty ice, containers and surfaces can increase the number of bacteria on seafood and speed spoilage. Contamination with food poisoning bacteria can cause illness when the seafood is eaten. Keeping seafood handling and storage equipment clean reduces bacterial contamination and slows spoilage.