Detection of fd born pathogens
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
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
, and eggs
The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:
Apply a sample of known antigen to a surface, often the well of a microtiter plate
. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve
used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
The plate wells or other surface are then coated with serum
samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
A concentrated solution of non-interacting protein, such as Bovine Serum Albumin
(BSA) or casein
, is added to all plate wells. This step is known as blocking
, because the serum proteins block non-specific adsorption of other proteins to the plate.
The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
The plate is washed to remove any unbound detection antibody. After this wash, only the antibody-antigen complexes remain attached to the well.
Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic signal.
View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical device.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
Prepare a surface to which a known quantity of capture antibody is bound.
Block any non specific binding sites on the surface.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.
Apply a detection antibody, with the same antigen specificity as the immobilized capture antibody.
Apply enzyme-linked secondary antibodies which are specific to the detection antibodies.
Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
Apply a chemical which is converted by the enzyme into a color or fluorescent signal.
Measure the absorbance or fluorescence of the plate wells to determine the presence and quantity of antigen.
The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the detection antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
Unlabeled antibody is incubated in the presence of its antigen.
These bound antibody/antigen complexes are then added to an antigen coated well.
The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
The secondary antibody
, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.http://en.wikipedia.org/wiki/Enzyme-linked_immunosorbent_assay
Detection of hazards:
- High Performance Liquid Chromatography
The Principles of High Pressure (Performance) Chromatography
Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. Different components of the sample are carried forward at different rates by the moving liquid phase, due to their differing interactions with the stationary and mobile phases. There are a number of different kinds of chromatography, which differ in the mobile and the stationary phase used.
In HPLC: The Mobile Phase is a solvent. This solvent is pumped under high pressure through a column.
The Stationary Phase is a finely divided solid held inside the column.
What is HPLC anyway?High Performance Liquid Chromatography (HPLC) is a chemistry based tool for quantifying and analyzing mixtures of chemical compounds.
What is HPLC used for?It's used to find the amount of a chemical compound within a mixture of other chemicals. An example would be to find out how much caffeine there is in the cup of coffee (or tea, or cola).
What I must do to analyze the sample?Dissolve the sample in a solvent (like water or alcohol), thus the term LIQUID chromatography.
How do I measure the sample amount?A detector measures response changes between the solvent itself, and the solvent & sample when passing through it. The electrical response is digitized and sent to a data system.
Comparison of HPLC over Gas Chromatography Less volatile and larger samples can be used with HPLC. It was discovered that better separation of the components of the mixture occurs if the particles in the stationary phase are very small. However, it was also found that if very small particles were used in the column, then the liquid passed very slowly through the column. Therefore, a pump is used to force the liquid through the column. This is not necessary in GC but a shorter column can be used in HPLC because the separation is more efficient.http://www.wesleylearning.ie/resources/science/chemistry/topics/instrumentation/hplc/principle.htm
Types of HPLC
Normal phase chromatography
Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separates analytes based on polarity. This method uses a polar stationary phase and a nonpolar mobile phase, and is used when the analyte of interest is fairly polar in nature. The polar analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increase in analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength not only depends on the functional groups in the analyte molecule, but also on steric factors and structural isomers are often resolved from one another. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes while more hydrophobic solvents tend to increase retention times. Particularly polar solvents in a mixture tend to deactivate the column by occupying the stationary phase surface. This is somewhat particular to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather than a soft layer on a surface)..
NP-HPLC had fallen out of favor in the 1970's with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HILIC
bonded phases which utilize a partition mechanism which provides reproducibility.
Reversed phase chromatography
Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and a moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. The retention time is therefore longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily. Retention time is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent. Reversed phase chromatography is so commonly used that it is not uncommon for it to be incorrectly referred to as "HPLC" without further specification.
RP-HPLC operates on the principle of hydrophobic interactions which result from repulsive forces between a relatively polar solvent, the relatively non-polar analyte, and the non-polar stationary phase. The driving force in the binding of the analyte to the stationary phase is the decrease in the area of the non-polar segment of the analyte molecule exposed to the solvent. This hydrophobic effect is dominated by the decrease in free energy
associated with the minimization of the ordered molecule-polar solvent interface. The hydrophobic effect is decreased by adding more non-polar solvent into the mobile phase. This shifts the partition coefficient such that the analyte spends some portion of time moving down the column in the mobile phase, eventually eluting from the column.
The characteristics of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a longer alkyl chain length results in a longer retention time because it increases the molecule's hydrophobicity. Very large molecules, however, can result in incomplete interaction between the large analyte surface and the alkyl chain. Retention time increases with hydrophobic surface area which is roughly inversely proportional to solute size. Branched chain compounds elute more rapidly than their corresponding isomers because the overall surface area is decreased.
Aside from mobile phase hydrophobicity, other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a linear increase in the surface tension of aqueous solutions, and because the entropy
of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. Another important component is pH
since this can change the hydrophobicity of the analyte. For this reason most methods use a buffering agent
, such as sodium phosphate
, to control the pH. An organic acid such as formic acid
or most commonly trifluoroacetic acid
is often added to the mobile phase. These serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. The effect varies depending on use but generally improve the chromatography.
Reversed phase columns are quite difficult to damage compared with normal silica columns, however, many reverse phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases
as these will destroy the underlying silica backbone. They can be used with aqueous acid but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column
is to inject a sample which is a mixture
of 2,2'- and 4,4'- bipyridine
. Because the 2,2'-bipy can chelate
the metal it is normal that when a metal ion
is present on the surface of the silica
the shape of the peak for the 2,2'-bipy will be distorted, tailing will be seen on this distorted peak.
Size exclusion chromatography
.Size exclusion chromatography
(SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of size. It is generally a low resolution
chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure
and quaternary structure
of purified proteins, and is the primary technique for determining the average molecular weight of natural and synthetic polymers
Ion exchange chromatography
In Ion-exchange chromatography, retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded. Some types of Ion Exchangers include: (1) Polystyrene resins- allows cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity. (2) Cellulose and dextran ion exchangers (gels)-These possess larger pore sizes and low charge densities making them suitable for protein separation.(3)Controlled-pore glass or porous silica.
In general, ion exchangers favor the binding of ions of higher charge and smaller radius.
An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. An increase in pH reduces the retention time in cation exchange while a decrease in pH reduces the retention time in anion exchange.
This form of chromatography is widely used in the following applications: In purifying water, preconcentration of trace components, Ligand-exchange chromatography, Ion-exchange chromatography of proteins, High-pH anion-exchange chromatography of carbohydrates and oligosaccharides, etc.
This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of these complexes involves the participation of common molecular forces such as the Van der Waal's interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.
Isocratic flow and gradient elution
With regard to the mobile phase, a composition that remains constant throughout the procedure is termed isocratic.
In contrast to this is the so called "gradient elution", which is a separation where the mobile phase changes its composition during a separation process. One example is a gradient in 20 min starting from 10% Methanol and ending up with 30% Methanol. Such a gradient can be increasing or decreasing. The benefit of gradient elution is that it helps speed up elution by allowing components that elute more quickly to come off the column under different conditions than components which are more readily retained by the column. By changing the composition of the solvent, components that are to be resolved can be selectively more or less associated with the mobile phase as a result at equilibrium they spend more time in the solvent and less in the stationary phase therefore they elute faster.
(GLC), or simply gas chromatography (GC), is a type of chromatography
in which the mobile phase is a carrier gas, usually an inert
gas such as helium
or an unreactive gas such as nitrogen
, and the stationary phase is a microscopic layer of liquid or polymer
on an inert solid support, inside glass or metal tubing, called a column. The instrument used to perform gas chromatographic separations is called a gas chromatograph (also: aerograph, gas separator).
A gas chromatograph is a chemical analysis instrument for separating chemicals
in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and identified electronically
. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, and the temperature.
In a GC analysis, a known volume of gaseous or liquid analyte
is injected into the "entrance" (head) of the column, usually using a microsyringe
(or, solid phase microextraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption
of the analyte molecules
either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption
, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time). A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column.
Thin Layer Chromatography (TLC) is a widely-used chromatography
technique used to separate chemical compounds 
. It involves a stationary phase consisting of a thin layer of adsorbent
material, usually silica gel
, aluminium oxide
, or cellulose
immobilised onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action
, separating the experimental solution. It can be used to determine the pigments a plant contains, to detect pesticides or insecticides in food, in forensics
to analyze the dye composition of fibers, or to identify compounds present in a given substance, among other uses. It is a quick, generic method for organic reaction monitoring.
TLC plates are made by mixing the adsorbent, such as silica gel
, with a small amount of inert
binder like calcium sulfate
(gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass
, thick aluminum foil, or plastic, and the resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC. Every type of chromatography contains a mobile phase and a stationary phase
The process is similar to paper chromatography
with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions
and for the qualitative analysis of reaction products.
A small spot of solution containing the sample is applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable solvent
, such as ethanol
, and placed in a sealed container. The solvent
moves up the plate by capillary action
and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds
in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the stationary phase. Results also vary depending on the solvent used. For example, if the solvent were a 90:10 mixture of hexane to ethyl acetate, then the solvent would be mostly nonpolar. This means that when analyzing the TLC, the nonpolar parts will have moved further up the plate. The polar compounds, in contrast, will not have moved as much. The reverse is true when using a solvent that is more polar than non-polar (10:90 hexane to ethyl acetate). With these solvents, the polar compounds will move higher up the plate, while the non-polar compounds will not move as much.
The appropriate solvent in context of Thin layer chromatography will be one which differs from the stationary phase material in polarity. If polar solvent is used to dissolve the sample and spot is applied over polar stationary phase TLC, the sample spot will grow radially due to capillary action, which is not advisable as one spot may mix with the other. Hence, to restrict the radial growth of sample-spot, the solvent used for dissolving samples in order to apply them on plates should be as non-polar or semi-polar as possible when the stationary phase is polar, and vice-versa.
Chromatogram of 10 essential oils
coloured with vanillin
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
Often a small amount of a fluorescent
compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a blacklight
(UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.Iodine
vapors are a general unspecific color reagent
Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate
Once visible, the Rf value of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants.http://en.wikipedia.org/wiki/Thin_layer_chromatographyAtomic absorption spectroscopy
(often called AA) - 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.
Atomic absorption spectroscopy (AAS) determines the presence of metals in liquid samples. Metals include Fe, Cu, Al, Pb, Ca, Zn, Cd and many more. It also measures the concentrations of metals in the samples. Typical concentrations range in the low mg/L range.
In their elemental form, metals will absorb ultraviolet light when they are excited by heat. Each metal has a characteristic wavelength that will be absorbed. The AAS instrument looks for a particular metal by focusing a beam of uv light at a specific wavelength through a flame and into a detector. The sample of interest is aspirated into the flame. If that metal is present in the sample, it will absorb some of the light, thus reducing its intensity. The instrument measures the change in intensity. A computer data system converts the change in intensity into an absorbance.
As concentration goes up, absorbance goes up. The researcher can construct a calibration curve by running standards of various concentrations on the AAS and observing the absorbances. In this lab, the computer data system will draw the curve for you! Then samples can be tested and measured against this curve.http://www.gmu.edu/departments/SRIF/tutorial/aas/aas.htm
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. Protein electrophoresis can also be performed on urine.
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.
Certain other diagnostic tests or prescription medications can affect the results of SPEP tests. The administration of a contrast dye used in some other tests may falsely elevate protein levels. Drugs that can alter results include aspirin, bicarbonates, chlorpromazine (Thorazine), corticosteroids, isoniazid (INH), and neomycin (Mycifradin).
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. For example, a decrease in albumin with a rise in the alpha2 globulin usually indicates an acute reaction of the type that occurs in infections, burns, stress, or heart attack. On the other hand, a slight decrease in albumin, with a slight increase in gammaglobulin, and a normal alpha2 globulin is more indicative of a chronic inflammatory condition, as might be seen in cirrhosis of the liver.
Protein electrophoresis is performed on urine samples to classify kidney disorders that cause protein loss. Here also certain band patterns are specific for disease. For example, the identification of a specific protein called the Bence Jones protein (by performing the Bence Jones protein test) during the procedure suggests multiple myeloma.http://www.healthatoz.com/healthatoz/Atoz/common/standard/transform.jsp?requestURI=/healthatoz/Atoz/ency/protein_electrophoresis.jsp
DNA electrophoresis is an analytical technique used to separate DNA
fragments by size. An electric field
forces the fragments to migrate through a gel
. DNA molecules normally migrate from negative to positive potential due to the net negative charge of the phosphate
backbone of the DNA chain. At the scale of the length of DNA molecules, the gel looks much like a random, intricate network. Longer molecules migrate more slowly because they are more easily 'trapped' in the network.
After the separation is completed, the fractions of DNA fragments of different length are often visualized using a fluorescent dye
specific for DNA, such as ethidium bromide
. The gel shows bands corresponding to different DNA molecules populations with different molecular weight. Fragment size is usually reported in "nucleotides", "base pairs" or "kb" (for 1000's of base pairs) depending upon whether single- or double-stranded DNA has been separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length.
The types of gel most commonly used for DNA electrophoresis are agarose
(for relatively long DNA molecules) and polyacrylamide
(for high resolution of short DNA molecules, for example in DNA sequencing
). Gels have conventionally been run in a "slab" format such as that shown in the figure, but capillary electrophoresis
has become important for applications such as high-throughput DNA sequencing. Electrophoresis techniques used in the assessment of DNA damage
include alkaline gel electrophoresis
and pulsed field gel electrophoresis
. The measurement and analysis are mostly done with a specialized gel analysis software
. Capillary electrophoresis results are typically displayed in a trace view called an electropherogram
The DNA strand is cut into smaller fragments using DNA endonuclease, then samples of the DNA solution (DNA sample and buffer) is placed in the wells of the gel, and allowed to run for some time (the less the voltage of the electophoresis, te longer time for the DNA sample to run through the gel, and this results in a more accurate separation). method for DNA electrophoresishttp://en.wikipedia.org/wiki/DNA_electrophoresis
Detection of aflatoxins
Cultural methods for detection of aflatoxins:
Aflatoxins present important food safely problems in both developed and developing countries. Contamination is monitored in developed countries using enzyme-linked immunusorbent assay (ELISA)- and high-performance liquid chromatography (HPLC)-based assays, both of which may be too expensive for routine use in many developing countries. There is a need for inexpensive alternative approaches to detect aflatoxins in lots of foods and feeds. Reviewed here are culture-based methods that determine if a sample is contaminated with aflatoxigenic fungi. These approaches include (1) blue fluorescence of aflatoxin B1, particularly when enhanced by including ß-cyclodextrin in the culture medium, (2) yellow pigment production, and (3) colour change on exposure to ammonium hydroxide vapour. The presence of aflatoxin B1 can be detected by its blue fluorescence, which is enhanced when the toxin complexes with the hydrophobic pocket of ß-cyclodextrin. The yellow pigment and ammonium hydroxide vapour tests are based on the production of yellow anthraquinone biosynthetic intermediates in the aflatoxin pathway. These compounds act as pH indicator dyes, which are more visible when they have turned red at alkaline pH. Because these tests are based on two different mechanisms, it has been possible to combine them into a single test. In a study of 517 A. flavus isolates from the Mississippi Delta, the combined assay reduced false positives for aflatoxigenicity to 0%, and false negatives to 7%. The increased predictive power of the combined cultural assay may enable its use for inexpensively identifying
potential aflatoxin contamination in feeds and foodshttp://www.cababstractsplus.org/google/abstract.asp?AcNo=20043182722HOW CAN IT BE DETECTED?
Since most samples do not contain a detectable amount of aflatoxin, there is a need for a method which correctly identifies the many negative samples with minimum expenditure of time and money. Such a method is known as the BLACK LIGHT TEST and UVP BLAK-RAY® lamps are recommended for this application. Refer to UVP Brochure 818.
The aflatoxins all have absorption maxima around 360nm with a molar absorptivity of about 20,000: the B toxins are named for their blue fluorescence (425nm) and the G toxins for their green-blue fluorescence (450nm). The B1 toxin is the most common, flowed by B2 toxin, while the G toxins are fairly rare. The fluorescence sensitivity of the G toxins is more than 10 times greater than that for the B toxins. The minimum detectable level is about 100 pgrams for the G toxins and 1 ngram for the B toxins.
Corn is inspected under the BLAK-RAY lamp for a characteristic bright greenish-yellow (BGY) fluorescence in broken and damaged kernels. The test takes 5 minutes or less. If the fluorescence is observed, aflatoxin may be present but not necessarily in appreciable or detectable levels. There are substances in corn and other food that fluoresce under long wave ultraviolet irradiation, but are not associated with aflatoxin. Many other fungi such as Aspergillus niger, various Penicillium species, Aspergillus repens and other species which do not produce aflatoxin may produce fluorescent harmless metabolites so that the fluorescence is not a specific indication of the presence of toxicogenic molds, although it may indicate that conditions have been favorable for growth of the toxicogenic molds.
Additionally, A. flavus, isolated from corn, has displayed a broad spectrum of aflatoxin production, ranging from no detectable yields to high levels (>100 ppm). A probable contributory factor to this is that the fluorescence in naturally contaminated corn is not stable and disappears in 4 to 6 weeks on continuous exposure to visible or ultraviolet radiation although the toxin does not disappear. Fresh samples mush therefore be taken.
The reliability of the method depends on the size of the sample taken for analysis and how it is taken. A sample mush be large enough to be representative of the entire lot of corn and must be taken from all parts of the lot–whether bin, truck or railroad car. One highly contaminated corn kernel may account for objectionable levels of aflatoxin in a 3000-kernel (2 lb.) sample. If only a 1 to 2 lb. sample is collected, the one highly contaminated kernel could be missed. Usually from 5 to 10 lb. samples are collected, but even larger ones would be better. If the sampling is not performed properly, the results obtained by this screening method could be of little value.
The black light test is an excellent screening test for possible presence of aflatoxin, but it does not give quantitative indication. Even with good technique, brightly fluorescing samples may contain less aflatoxin than weakly fluorescing samples. Because of this, additional confirmatory and quantitative measurements are needed to be applied to those samples that reacted positively to the black light test. Non-fluorescing samples need not be subjected to this.FURTHER SCREENING
Since the black light test is only a preliminary confirmatory test, another screening test is used in feed mills immediately following the black light test. This test involves the use of a small chromatographic column (mini-column) in the final separation and detection step of the method.
If the black light test gives a positive answer, the 10 lb. sample of cracked corn is ground to pass a twenty-mesh screen and the mixed thoroughly. A 50 g representative subsample of the 10 lb. of finely ground corn is used for the mini-column test. The final separation step of the mini-column method involves transferring 3 ml of chloroform extract to the top of a mini-column. After the chloroform drains into the column, 3 ml of an elution solvent are passed through the mini-column.
The mini-column is then inspected in the dark by shining a long wave black light on it. This, again, should be one of the BLAK-RAY lamps. If a bluish-green fluorescent band is detected at the proper height in the mini-column, the sample is judged to be positive to aflatoxin. In this instance, the property detected by the test is the bluish-green fluorescence.
This test, like the black light test, is a good screening test because it gives few false negative answers. However, this test also has the characteristic of giving only a few false positive answers. This makes it a better screening test, from a scientific viewpoint, than the black light test; however, it is better cost wise to run the two tests one after the other.
After the mini-column test is completed, a judgment can be made as to whether or not a shipment of corn will be accepted by a feed mill. The changes of accepting a truckload of corn that contains more than the 20 ppb aflatoxin is slight, as is the chance of rejected a truckload of corn that does not contain detectable aflatoxin.
If further confirmation is required, a subsample of the ground sample of corn can be sent to a chemical laboratory, or the extract remaining from the mini-column test can be used for a simple confirmatory test.
In this test, a portion of the chloroform extract not used for the mini-column test is evaporated to a small volume and some of this concentrated extract is spotted on a thin layer plate. A small amount of trifluoroacetic acid (TFA) is placed on top of the spot where the sample extract was placed. If aflatoxin is present in the sample spot on the thin layer plate, it will be derivatized to a water adduct of the parent aflatoxin compound. After thin layer chromatography is performed, this plate is inspected in the dark under a long wave black light. If a bluish-green fluorescent spot is detected on the thin layer plate at the same height as a reference spot known to be the derivative of aflatoxin formed by the TFA reaction, this confirmatory test (the TFA test) is positive and the concentration probably greater than 5 to 10 ppb.QUALITATIVE TESTS
Although the TFA test is a confirmatory test, it is not essentially different from the mini-column test. Both are qualitative tests, that is, both test for qualities or properties of aflatoxin. The final property of aflatoxin involved in the mini-column test is the property fluorescing bluish-green under a long wave black light.
The TFA test involves three additional properties of aflatoxin. One of the properties involved is that aflatoxin will form a derivative in the presence of trifluoroacetic acid while on a thin layer plate at room temperature. Another property is that this derivative will move to a certain height on the thin layer plate using a certain developing solvent. A third property is that this derivative will fluoresce bluish-green under a long wave black light.
However, even if this test also is positive, we are still not 100% certain that aflatoxin has been detected. At this point, the degree of certainty is probably over 99%.
If we desire a further confirmation that aflatoxin is in the sample, a small amount of the underivatized compound can be isolated on a thin layer plate and extracted. This small amount of sample can then be inserted into the entrance of a mass spectrometer. The mass spectrometer is an instrument, which will break the aflatoxin into many fragments. The original mass of the aflatoxin molecule, plus that of each of the fragments produced, will be indicated by the output of the instrument.
The pattern of the various masses is characteristic of the particular aflatoxin involved. If the pattern of the chemical suspected to be aflatoxin is identical with the pattern given by a know aflatoxin, we are about 99.99% certain that the suspect chemical is aflatoxin