protein electrophoresis

Definition
Electrophoresis is a technique used to separate different elements (fractions) of a blood sample into individual components. Serum protein electrophoresis (SPEP) is a screening test that measures the major blood proteins by separating them into five distinct fractions: albumin, alpha1, alpha2, beta, and gamma proteins.

Purpose
Protein electrophoresis is used to evaluate, diagnose, and monitor a variety of diseases and conditions. It can be used for these purposes because the levels of different blood proteins rise or fall in response to such disorders as cancer, intestinal or kidney protein-wasting syndromes, disorders of the immune system, liver dysfunction, impaired nutrition, and chronic fluid-retaining conditions.

Description
Proteins are major components of muscle, enzymes, hormones, hemoglobin, and other body tissues. Proteins are composed of elements that can be separated from one another by several different techniques: chemical methods, ultracentrifuge, or electrophoresis. There are two major types of electrophoresis: protein electrophoresis and immunoelectrophoresis. Immunoelectrophoresis is used to assess the blood levels of specific types of proteins called immunoglobulins. An immunoelectrophoresis test is usually ordered if a SPEP test has a "spike," or rise, at the immunoglobulin level. Protein electrophoresis is used to determine the total amount of protein in the blood, and to establish the levels of other types of proteins called albumin, alpha1 globulin, alpha2 globulin, and beta-globulin.

Electrophoretic measurement of proteins
All proteins have an electrical charge. The SPEP test is designed to make use of this characteristic. There is some difference in method, but basically the sample is placed in or on a special medium (e.g., a gel), and an electric current is applied to the gel. The protein particles move through the gel according to the strength of their electrical charges, forming bands or zones. An instrument called a densitometer measures these bands, which can be identified and associated with specific diseases

enzyme immunoassay

The 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. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. 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 a mild detergent solution 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. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity. In simple terms, an unknown amount of antigen in a sample is immobilized on a surface. One then washes a particular antibody over the surface. This antibody is linked to an enzyme that visibly reacts when activated, say by light hitting it in the case of a fluorescent enzyme; the brightness of the fluorescence would then tell you how much antigen is in your sample.
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test[1] or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.

Atomic absorption spectroscopy

This method commonly uses a pre-burner nebulizer (or nebulizing chamber) to create a sample mist and a slot-shaped burner which gives a longer pathlength flame. The temperature of the flame is low enough that the flame itself does not excite sample atoms from their ground state. The nebulizer and flame are used to desolvate and atomize the sample, but the excitation of the analyte atoms is done by the use of lamps shining through the flame at various wavelengths for each type of analyte. In AA, the amount of light absorbed after going through the flame determines the amount of analyte in the sample. A graphite furnace for heating the sample to desolvate and atomize is commonly used for greater sensitivity. The graphite furnace method can also analyze some solid or slurry samples. Because of its good sensitivity and selectivity, it is still a commonly used method of analysis for certain trace elements in aqueous (and other liquid) samples.

Chromatography

Chromatography is method of separating mixtures and identifying their components i.e. it's a separation method that exploits the differences in partitioning behavior of analytes between a mobile phase and a stationary phase to separate components in a mixture. Components of a mixture may be interacting with the stationary phase based on charge (ion-ion-interactions, ion-dipole-interactions), van der Waals' forces, relative solubility or adsorption (hydrophobic interactions, specific affinity). There are two theories of chromatography, the plate and rate theories

Gas chromatography

Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see below) .
Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silcone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.

Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC).
In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.
Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography.

Polymerase chain reaction

PCR's power lies in its ability to easily isolate particular regions of DNA sequence from whole genomic material. Many techniques need a pool of DNA molecules isolated from a particular DNA fragment, and the use of PCR has enabled these techniques more widespread in usage. Because PCR also amplifies the isolated region, the techniques are more powerful, applicable to samples otherwise too small for analysis.

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.[2]
PCR, as currently practiced, requires several basic components [3]. These components are:
DNA template that contains the region of the DNA fragment to be amplified
One or more primers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified.
a DNA polymerase (e.g. Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C), used to synthesize a DNA copy of the region to be amplified
Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
Buffer solution, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase
Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis [4]
Monovalent cation potassium ions
The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a reaction volume typically of 15-100 μl, that are inserted into a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. Most thermal cyclers have heated lids to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation.

mycotoxins in GM foods

Genetic modification (GM) has been promoted as a means of preventing mycotoxin contamination. Insect-resistant crops are genetically modified with genetic material from a naturally occurring soil-borne bacterium, Bacillus thuringiensis that produces a protein that is toxic to certain insect pests. BT crops have been shown to be non toxin to humans and safe for consumption, it is both beneficial for the environment and for farmers.

The fumonisins, a family of food-borne carcinogenic mycotoxins, are isolated from cultures of F. verticillioides. The fungus Fusarium verticillioides is one of the most prevalent seed-borne fungi associated with maize intended for human and animal consumption throughout the world. They cause liver disease and cancer in rodents and are considered possible risk factors for cancer in man. The recommended daily intake of fumonisins is 0.5μg per person a day

The mycotoxin aflatoxin B1 (AFB1), which is produced by the fungus Aspergillus flavus that grows on peanuts before or after harvesting and under poor storage conditions. AFB1 is classified as a carcinogen - a substance that can cause cancer in humans. The mycotoxin aflatoxin B1 (AFB1), which is produced by the fungus Aspergillus flavus that grows on peanuts before or after harvesting and under poor storage conditions. AFB1 is classified as a carcinogen - a substance that can cause cancer in humans. The FDA recommended intake of aflatoxin is 20 ppb for humans, immature animals (including poultry) and all dairy animals, 100 ppb for breeding beef cattle, swine and mature poultry, 200 ppb for finishing swine (100 pounds and up) and 300 ppb for feeder cattle

Another mycotoxin is patulin - this is a toxic secondary metabolite that is produced by a number of fungi, most important of which is Penicillium expansum. This fungus is a well-known post-harvest pathogen that causes ‘blue mould rot’ or ‘soft’ rots' in apples. Patulin has been shown to possess mutagenic properties (can cause damage to the genetic material of cells), and to cause immunotoxic, neurotoxic and gastro-intestinal effects in rodents. Recommended specifications are that patulin levels should not exceed 50 parts per billion (μg/L) in products intended for human consumption.

Immunoassays, PCR```

Methods for the identification of GM organism in food can be divided into 3 categories. In the first category are nucleotide-based amplification methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), fingerprinting techniques (such as RFLP, AFLP, and RAPD), probe hybridization, “self-sustained sequence replication” (3SR), and “Q replicase amplification”. The second category involves protein-based methods including one dimensional SDS gel electrophoresis, two-dimensional SDS gel electrophoresis, Western-blot analysis and ELISA(enzyme-linked immunoabsorbant assay). The third category is based on the detection of enzymatic activities.
Every detection method has its own specificity and limitations. The detection using an enzymatic activity method is not recommended for processed foods, where proteins may be denaturized.



PCR:
The methods based on PCR are not suitable for detection of highly processed foods because DNA fragments in foods could be broken into pieces. Among the 3 categories, PCR is the most popular method used worldwide. The key elements in the PCR process are as follows:
“primers”—small DNA molecules whose sequences correspond to the target sequence.
A heat stable DNA polymerase—typically Taq polymerase, which synthesizes new copies of the target sequence in a manner that is dependent upon the interaction of primers with these target sequences.
A thermocycler—an apparatus that can be programmed to carry the contents of the PCR reaction vessels through multiple, precisely controlled temperature cycles.



Immunoassays:
Immunoassays are ideal techniques for quantitative and qualitative detection of a variety of proteins in complex material when the target analyte is known. An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets Immunoassays utilizing high affinity polyclonal antibodies resulted in high throughput, sensitive and specific in vitro analytical procedures.

(An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]

Near infrared spectroscopy:
NIR spectroscopy relies on the relationship between a sample’s absorbance of incident NIR radiation, and the concentration of absorbing species within the sample. When NIR radiation impinges on a sample, certain molecules with the sample will vibrate depending on their mass, chemical bonding structure, and the wavelength of the incident radiation. This relationship makes vibrational spectra useful for gaining insight into the molecular structure of a compound.

Hybridization
It comprises amplifying transgenes of GMO by biotin-labeled primer sets, hybridizing the amplified products with colored bead-labeled probes, and detecting the hybrids.