EIA also known as Enzyme -Linked ImmunoSorbent Assay function based on the detection and measurement of primary antibody-antigen binding reaction.
Relevant antibody bound to a solid phrase (polystyrene microtiter plates), heat-treated broth culture (from food) is then added to the well. Unbound antigen washed away; antibody has a specific shape, it will only bind to one particular kind of antigen due to its key-and-lock principle. And only when antigens are bounded to the antibody on the polystyrene microtiter plates, will there be positive results. After the antigen is immobilized the enzyme labelled antibody (conjugate), sometimes known as detection antibody is then 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. And incubated, after incubation, unbound conjugate washed away. Specific substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic signal is subsequently added to show the reaction via enzymatic reactions. Followed by incubation at room temperature; to allow enzymatic reactions to take place. Reaction ceased by addition of sulfuric acid. Results read through photometer or other optical devices at specific wavelength.
ELISA tests can be categorized into indirect, sandwich or capture. Indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample, whether it is from the serum of an immunized animal or the cell supernatant from growing hybridoma clones. Sandwich ELISA is used to determine the antigen concentration in unknown samples. Competitive ELISA is used when two “matched pair” antibodies are not available for experiment target. Different ELISA tests have different procedures however, the principles behind these tests are similar.
Tuesday, July 31, 2007
DNA Probe
Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all living organisms. Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from DNA. As DNA and RNA of organisms are specifically different between each other, by identifying the DNA and RNA of organisms can then be used to isolate and identify. Foodborne pathogen can be detected when DNA or RNA of the food samples are complementary to the nucleotide sequence in the probe. If DNA or RNA of food samples match that of the DNA probe, a complex is formed and thus a positive results.
New DNA probe assays for detection of Salmonella, Listeria, E. coli and Staphyococcus aureus use non-isotopically labelled DNA probe to detect specific ribosomal RNA targets of organisms. Test samples will first undergo lysis, using alkaline or enzymatic reagents which cause the cells to burst and die. Probes are then added to the test samples to form probe-target complexes; in solution form. These complexes formed and hybridized will be captured onto polystyrene sticks to separate the desired components from the unwanted cellular and sample debris. Hybridization is a process of combining complementary, single-stranded nucleic acids into a single molecule. The single molecule thus produced would then be detected after hybridization by its intrinsic properties (e.g., fluorescence) or through recognition by a specific antibody. If detection is by intrinsic properties, the polystyrene sticks will be incubated with solution containing an anti-fluroescein antibody-horseradish peroxidase conjugate and a mixture of tetramethyl benzidine/hydrogen peroxide. After reaction ceased, results can then be obtained by determining the absorbance of samples at a specific wavelength (usually 450nm) using photometer instrument or other optical devices.
New DNA probe assays for detection of Salmonella, Listeria, E. coli and Staphyococcus aureus use non-isotopically labelled DNA probe to detect specific ribosomal RNA targets of organisms. Test samples will first undergo lysis, using alkaline or enzymatic reagents which cause the cells to burst and die. Probes are then added to the test samples to form probe-target complexes; in solution form. These complexes formed and hybridized will be captured onto polystyrene sticks to separate the desired components from the unwanted cellular and sample debris. Hybridization is a process of combining complementary, single-stranded nucleic acids into a single molecule. The single molecule thus produced would then be detected after hybridization by its intrinsic properties (e.g., fluorescence) or through recognition by a specific antibody. If detection is by intrinsic properties, the polystyrene sticks will be incubated with solution containing an anti-fluroescein antibody-horseradish peroxidase conjugate and a mixture of tetramethyl benzidine/hydrogen peroxide. After reaction ceased, results can then be obtained by determining the absorbance of samples at a specific wavelength (usually 450nm) using photometer instrument or other optical devices.
Friday, July 20, 2007
HPLC - Principles
The basic operating principle of HPLC is to force the analyte through a column of the stationary phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combinations of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent.
A further refinement to HPLC has been to vary the mobile phase composition during the analysis, this is known as gradient elution. A normal gradient for reverse phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol; whereas the more hydrophilic compounds will elute under conditions of relatively low methanol. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best separation of peaks.
A further refinement to HPLC has been to vary the mobile phase composition during the analysis, this is known as gradient elution. A normal gradient for reverse phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol; whereas the more hydrophilic compounds will elute under conditions of relatively low methanol. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best separation of peaks.
GC/MS
Gas Chromatograph/Mass Spectrometer is also known informally as GC/MS.
A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure. There are many types of mass spectrometers and sample introduction techniques which allow a wide range of analyses. This discussion will focus on mass spectrometry as it's used in the powerful and widely used method of coupling Gas Chromatography (GC) with Mass Spectrometry (MS).
A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure. There are many types of mass spectrometers and sample introduction techniques which allow a wide range of analyses. This discussion will focus on mass spectrometry as it's used in the powerful and widely used method of coupling Gas Chromatography (GC) with Mass Spectrometry (MS).
Mass Spectrometer
MS measures the mass-to-change ratio (m/z) of ions that have been produced from the sample.
Sample -> Inlet -> ionization source -> analyzer -> Ion detector -> data system
From ionization source to ion detector, it's taken place in vaccum system.
Unionized molecules and fragments are pumped out of the ionization source. Ions are passed into an analyzer where they are separated according to their mass-to-change ratio. Ion strike an ion detector, where they produce an electrical signal that is recorded and plotted by data system.
The stages within the mass spectrometer are:
1. Producing ions from the sample
2. Separating ions of differing masses
3. Detecting the number of ions of each mass produced
4. Collecting the data and generating the mass spectrum
The technique has several applications, including:
* identifying unknown compounds by the mass of the compound molecules or their fragments
* determining the isotopic composition of elements in a compound
* determining the structure of a compound by observing its fragmentation
* quantifying the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative)
* studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum)
* determining other physical, chemical, or even biological properties of compounds with a variety of other approaches
Sample -> Inlet -> ionization source -> analyzer -> Ion detector -> data system
From ionization source to ion detector, it's taken place in vaccum system.
Unionized molecules and fragments are pumped out of the ionization source. Ions are passed into an analyzer where they are separated according to their mass-to-change ratio. Ion strike an ion detector, where they produce an electrical signal that is recorded and plotted by data system.
The stages within the mass spectrometer are:
1. Producing ions from the sample
2. Separating ions of differing masses
3. Detecting the number of ions of each mass produced
4. Collecting the data and generating the mass spectrum
The technique has several applications, including:
* identifying unknown compounds by the mass of the compound molecules or their fragments
* determining the isotopic composition of elements in a compound
* determining the structure of a compound by observing its fragmentation
* quantifying the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative)
* studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum)
* determining other physical, chemical, or even biological properties of compounds with a variety of other approaches
Tuesday, July 10, 2007
Toxin Detection
Toxic Metals
Rapid microwave digestion and acid digestion systems, and analysis using atomic absorption spectrophotometers and inductively coupled plasma-mass spectrometer
Polychlorinated Biphenyls (PCBs)
Gas chromatograph (GC)
Chloropropanols
Gas chromatograph / mass spectrometer (GC/MS)
Acrylamide
Liquid chromatograph / mass spectrometer / mass spectrometer (LC/MS/MS)
antibiotics, growth promotants and other veterinary drugs
Microbial inhibition assay for antibiotics
Enzyme-linked immunosorbent assay for beta-agonists, antibiotics and growth promotants
High performance liquid chromatography or LC/MS/MS for nitrofurans, coccidiostats and other antibiotics
Rapid microwave digestion and acid digestion systems, and analysis using atomic absorption spectrophotometers and inductively coupled plasma-mass spectrometer
Polychlorinated Biphenyls (PCBs)
Gas chromatograph (GC)
Chloropropanols
Gas chromatograph / mass spectrometer (GC/MS)
Acrylamide
Liquid chromatograph / mass spectrometer / mass spectrometer (LC/MS/MS)
antibiotics, growth promotants and other veterinary drugs
Microbial inhibition assay for antibiotics
Enzyme-linked immunosorbent assay for beta-agonists, antibiotics and growth promotants
High performance liquid chromatography or LC/MS/MS for nitrofurans, coccidiostats and other antibiotics
Saturday, July 7, 2007
Local Authority on GM Food
GMAC - Genetic Modification Advisory Committee
(Information adapted from www.gmac.gov.sg)
Roles & Responsibility
The Genetic Modification Advisory Committee was established in Singapore in April 1999 to oversee and advise on the research and development, production, use and handling of Genetically Modified Organisms (GMOs) in Singapore.
The objective of this committee was to ensure public safety while allowing for the commercial use of GMOs and GMO-derived products by companies and research institutions, in compliance with international standards.
Flow Chart for Evaluation, Approval and Registration of Genetically Modified Organisms (GMOs) Related to Agriculture
http://gmac.gov.sg/guidelines/agriculture_appendix_3.html
(Information adapted from www.gmac.gov.sg)
Roles & Responsibility
The Genetic Modification Advisory Committee was established in Singapore in April 1999 to oversee and advise on the research and development, production, use and handling of Genetically Modified Organisms (GMOs) in Singapore.
The objective of this committee was to ensure public safety while allowing for the commercial use of GMOs and GMO-derived products by companies and research institutions, in compliance with international standards.
Flow Chart for Evaluation, Approval and Registration of Genetically Modified Organisms (GMOs) Related to Agriculture
http://gmac.gov.sg/guidelines/agriculture_appendix_3.html
Thursday, July 5, 2007
ADI
What is Acceptable Daily Intake?
The Acceptable Daily Intake (ADI) is an estimate by JECFA of the amount of a food additive,expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk(standard man - 60 Kg) (WHO Environmental Health Criteria document N° 70, Principles for the Safety Assessment of food Additives and Contaminants in Food, Geneva, 1987). The ADI is expressed in milligrams of the additive per kilogram of body weight.
For this purpose, "without appreciable risk" is taken to mean the practical certainty that injury will not result even after a life-time's exposure (Report of the 1975 JMPR, TRS 592, WHO, 1976). The ADI is established over lifetime. A body weight of 60 kg is usually taken to represent the average weight of the population (Report of the 1988 JECFA , TRS 776 sec. 2.2.3. WHO, 1989). However, in some countries, and especially in the developing ones, a 50 kg body weight would better represent the average body weight of the population.
How To Establish ADI?
Before discussing different approaches used in estimating food additive intake, the methods of establishing an ADI need to be reviewed.
Groups of animals (e.g. rats) are given daily diets containing different levels of the additive under examination. For example, levels of the additives in the diet could be: 0.1%, 1%, 2%, 5%. If a toxic effect is found at the 2% level and a "no toxic effect" at 1% level, the 1% level (expressed in mg/kg body weight) will be
the "no-observed-effect level", and it is from this level that the extrapolation to humans is done. In this case, the no-observed-effect level lies between the 1% and 2% levels, and if no toxicological evaluations are done at intermediary levels (1.25%, 1.50%, 1.75%) the choice of the 1% level as the no-observed-effect level introduces
already a first safety factor. The extrapolation from the no-observed-effect level to an ADI is often done by using a safety factor of 100 (10 x 10) which assumes that humans are 10 times more sensitive than experimental animals and that there
is a 10-fold variation in sensitivity within the human population. This safety factor of 100 is based on the experience and common sense of toxicologists and therefore cannot be compared to a physical value such a-s the boiling point of a pure substance. More information regarding the no-observed-effect level and the use of safety factors can be found in "Principles for the Safety Assessment of Food Additives and contaminants in Food".(Environmental Health Criteria No 70, WHO, Geneva 1987, p. 77-79).
Estimations of intake may be sequentially calculated starting with the simplest TMDI and proceeding to more refined EDI if necessary. When precise data on consumption of foodstuff exist, they should be used. When such precise data do not exist, approximations can be adequate to support a safe use. A hypothetical figure based upon extreme theoretical cases such as the TMDI can give adequate assurance of safety in use if such figure is lower than the ADI. However, if the ADI is exceeded, using this approach, before a decision is made a search would have to be made for data which approximate the actual intake (the TMDI can be improved by taking into account intake of special population groups).
What is TMDI?
The Theoretical Maximum Daily Intake (TMDI) is calculated by multiplying the average per capita daily food consumption for each foodstuff or food group by the legal maximum use level of the additive established by Codex standards or by national regulations and by summing up the figures.
The TMDI gives only a rough indication of the dietary intake of a food additive since it does not take into consideration the food habits of special populations groups, and it assumes that:
(a) all foods in which an additive is permitted contain that additive;
(b) the additive is always present at the maximum permitted level;
(c) the foods in question containing the additive are consumed by people each day of their lives at the average per capita level;
(d) the additive does not undergo a decrease in level as a result of cooking or processing techniques;
(e) all foods permitted to contain the additive are ingested and nothing is discarded.
The Acceptable Daily Intake (ADI) is an estimate by JECFA of the amount of a food additive,expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk(standard man - 60 Kg) (WHO Environmental Health Criteria document N° 70, Principles for the Safety Assessment of food Additives and Contaminants in Food, Geneva, 1987). The ADI is expressed in milligrams of the additive per kilogram of body weight.
For this purpose, "without appreciable risk" is taken to mean the practical certainty that injury will not result even after a life-time's exposure (Report of the 1975 JMPR, TRS 592, WHO, 1976). The ADI is established over lifetime. A body weight of 60 kg is usually taken to represent the average weight of the population (Report of the 1988 JECFA , TRS 776 sec. 2.2.3. WHO, 1989). However, in some countries, and especially in the developing ones, a 50 kg body weight would better represent the average body weight of the population.
How To Establish ADI?
Before discussing different approaches used in estimating food additive intake, the methods of establishing an ADI need to be reviewed.
Groups of animals (e.g. rats) are given daily diets containing different levels of the additive under examination. For example, levels of the additives in the diet could be: 0.1%, 1%, 2%, 5%. If a toxic effect is found at the 2% level and a "no toxic effect" at 1% level, the 1% level (expressed in mg/kg body weight) will be
the "no-observed-effect level", and it is from this level that the extrapolation to humans is done. In this case, the no-observed-effect level lies between the 1% and 2% levels, and if no toxicological evaluations are done at intermediary levels (1.25%, 1.50%, 1.75%) the choice of the 1% level as the no-observed-effect level introduces
already a first safety factor. The extrapolation from the no-observed-effect level to an ADI is often done by using a safety factor of 100 (10 x 10) which assumes that humans are 10 times more sensitive than experimental animals and that there
is a 10-fold variation in sensitivity within the human population. This safety factor of 100 is based on the experience and common sense of toxicologists and therefore cannot be compared to a physical value such a-s the boiling point of a pure substance. More information regarding the no-observed-effect level and the use of safety factors can be found in "Principles for the Safety Assessment of Food Additives and contaminants in Food".(Environmental Health Criteria No 70, WHO, Geneva 1987, p. 77-79).
Estimations of intake may be sequentially calculated starting with the simplest TMDI and proceeding to more refined EDI if necessary. When precise data on consumption of foodstuff exist, they should be used. When such precise data do not exist, approximations can be adequate to support a safe use. A hypothetical figure based upon extreme theoretical cases such as the TMDI can give adequate assurance of safety in use if such figure is lower than the ADI. However, if the ADI is exceeded, using this approach, before a decision is made a search would have to be made for data which approximate the actual intake (the TMDI can be improved by taking into account intake of special population groups).
What is TMDI?
The Theoretical Maximum Daily Intake (TMDI) is calculated by multiplying the average per capita daily food consumption for each foodstuff or food group by the legal maximum use level of the additive established by Codex standards or by national regulations and by summing up the figures.
The TMDI gives only a rough indication of the dietary intake of a food additive since it does not take into consideration the food habits of special populations groups, and it assumes that:
(a) all foods in which an additive is permitted contain that additive;
(b) the additive is always present at the maximum permitted level;
(c) the foods in question containing the additive are consumed by people each day of their lives at the average per capita level;
(d) the additive does not undergo a decrease in level as a result of cooking or processing techniques;
(e) all foods permitted to contain the additive are ingested and nothing is discarded.
Subscribe to:
Posts (Atom)