Questions and Answers
What is the purpose of OPD (o-phenylenediamine dihydrochloride) in an ELISA test?
To detect HRP (Horseradish Peroxidase) and turn amber.
What is the effect of adding sulfuric or phosphoric acid to TMB (3,3',5,5'-tetramethylbenzidine) in an ELISA test?
It turns yellow.
What is the color change observed when ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) detects HRP in an ELISA test?
It turns green.
What is the application of ELISA in the food industry?
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What is the primary reason for using a transparent gel in gel electrophoresis?
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What is one of the serological blood tests that ELISA is used for?
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What is the purpose of ELISA in toxicology?
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Why is it important for the experimenter to be able to adjust the pore size of the gel?
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What is one of the diseases that ELISA is used to detect?
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What are the two dominant compounds used for gel electrophoresis?
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What is PNPP (p-Nitrophenyl Phosphate, Disodium Salt) used to detect in an ELISA test?
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How is the three-dimensional network of polyacrylamide gel formed?
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How can the pore size of polyacrylamide gels be adjusted?
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What type of molecules are typically electrophoresed using polyacrylamide gels?
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How is the agarose gel formed?
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How can the pore size of agarose gels be controlled?
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What happens to the antibodies in a traditional ELISA test upon addition and washing?
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What is the purpose of the Scavenger container in Reverse ELISA?
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What is the advantage of Reverse ELISA over traditional ELISA?
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What device is used to detect the tagged and bound antibodies in Reverse ELISA?
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What is the significance of the sufficient incubation period in Reverse ELISA?
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What is the role of enzymatic markers in ELISA assays?
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In what type of setting is Reverse ELISA commonly used?
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What is the difference between traditional ELISA and Reverse ELISA in terms of test format?
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Why is it necessary to increase the concentration of magnesium with high concentrations of dNTPs?
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What are the two essential roles of dNTPs in DNA synthesis?
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What is the typical final concentration of dNTPs in a PCR reaction?
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What is the relationship between the number of cycles and the abundance of DNA matrix in PCR?
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What is the purpose of ethidium bromide staining in PCR product detection?
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What is the range of primer concentrations used in a PCR reaction?
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What is the maximum quantity of template DNA that can be used in a PCR reaction?
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What type of electrophoresis is used in automated systems for PCR product detection?
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What is the typical range of Taq polymerase units used per sample in a PCR reaction?
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What is required for PCR products to be detected by a laser diode in automated systems?
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How does the duration of the temperature cycles depend on the size of the sequence of interest?
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What is the dynamic range of quantification in real-time PCR assays?
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What is the significance of real-time PCR technology in molecular diagnostics?
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Why is it important to minimize the duration of the temperature cycles?
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What type of DNA fragments are often visible close to the migration front in agarose gel electrophoresis?
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What is the result of nonspecific DNA fragments being amplified in PCR?
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Study Notes
ELISA (Enzyme-Linked Immunosorbent Assay)
- A type of assay that detects and measures the presence of antibodies or antigens in a sample
- Can be used to evaluate the presence of antigen or antibody in a sample
- Applications include:
- Determining serum antibody concentrations (e.g., HIV test, West Nile virus)
- Detecting potential food allergens (e.g., milk, peanuts, walnuts, almonds, eggs)
- Serological blood test for coeliac disease
- Rapid presumptive screen for certain classes of drugs
- Detection of various diseases (e.g., dengue, malaria, Chagas disease, Johne's disease)
- In vitro diagnostics in medical laboratories
- Detection of specific antibodies (e.g., Mycobacterium, rotavirus, hepatitis B and C, enterotoxin of E.coli, HIV, SARS-CoV-2)
Reverse ELISA
- A type of ELISA that does not use traditional wells
- Antigens are suspended in the test fluid
- Steps:
- Incubate unlabeled antibody with its antigen (sample)
- Allow antibodies to bind to antigens
- Pass the sample through a Scavenger container with "Scavenger Antigens" bound to its surface
- Allow Scavenger Antigens to bind to excess antibodies
- Pass the sample through a detector (e.g., flow cytometer) to measure the response
- Allows for multiple antigens to be tagged and counted simultaneously
- Can identify specific strains of bacteria using different color tags
Enzymatic Markers
- Substances used in ELISA assays to measure the results
- Examples:
- OPD (o-phenylenediamine dihydrochloride) turns amber to detect HRP (Horseradish Peroxidase)
- TMB (3,3',5,5'-tetramethylbenzidine) turns blue when detecting HRP and turns yellow after adding sulfuric or phosphoric acid
- ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) turns green when detecting HRP
- PNPP (p-Nitrophenyl Phosphate, Disodium Salt) turns yellow when detecting alkaline phosphatase
Electrophoresis
- A laboratory technique used to separate and analyze mixtures of ions (e.g., proteins and nucleic acids)
- Two common compounds used for gel electrophoresis:
- Polyacrylamide: provides smaller pores, used for electrophoresis of proteins and small nucleic acids
- Agarose: provides larger pores, used for electrophoresis of large nucleic acids
PCR (Polymerase Chain Reaction)
- A laboratory technique used to amplify specific DNA sequences
- Steps:
- Template DNA: extract from any organism or complex biological material
- Primer concentration: 10-50 pmol per sample
- dNTPs (deoxyribonucleoside triphosphates): provide energy and nucleotides for DNA synthesis
- Matrix DNA: from any organism, not too degraded, and not contaminated with inhibitors
- Taq polymerase: 1-3 units per sample
- Temperature cycles: denaturation, hybridization, and elongation, with durations and number of cycles depending on the size of the sequence of interest
PCR Product Detection and Analysis
- Product: one or more amplified DNA fragments
- Detection and analysis:
- Agarose gel electrophoresis (or acrylamide)
- Ethidium bromide staining
- Ultraviolet transillumination (280-320 nm) to visualize the products
- Fragment analyzer: uses capillary electrophoresis, detection by laser diode, only possible with primers coupled to fluorochromes
Real-time Quantitative PCR (RT-PCR)
- A type of PCR that allows sensitive, specific, and reproducible quantification of mRNA
- Characteristics:
- Wide dynamic range of quantification (7-8 logarithmic decades)
- High technical sensitivity
- Applications:
- Molecular diagnostics
- High-throughput, automated technology with lower turnaround times
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Assess your knowledge of ELISA, a type of assay that detects and measures antibodies or antigens in a sample, and its various applications in medical testing.