ELISA Assay Applications
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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?

<p>Detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.</p> Signup and view all the answers

What is the primary reason for using a transparent gel in gel electrophoresis?

<p>So that separated ions can be visualized by staining procedures</p> Signup and view all the answers

What is one of the serological blood tests that ELISA is used for?

<p>Coeliac disease.</p> Signup and view all the answers

What is the purpose of ELISA in toxicology?

<p>As a rapid presumptive screen for certain classes of drugs.</p> Signup and view all the answers

Why is it important for the experimenter to be able to adjust the pore size of the gel?

<p>To accommodate the broad size range of molecules studied in molecular biology</p> Signup and view all the answers

What is one of the diseases that ELISA is used to detect?

<p>Dengue.</p> Signup and view all the answers

What are the two dominant compounds used for gel electrophoresis?

<p>Polyacrylamide and agarose</p> Signup and view all the answers

What is PNPP (p-Nitrophenyl Phosphate, Disodium Salt) used to detect in an ELISA test?

<p>Alkaline phosphatase.</p> Signup and view all the answers

How is the three-dimensional network of polyacrylamide gel formed?

<p>By the incorporation of N,N'-methylenebisacrylamide into the polymerizing chains, resulting in crosslinks between the long chains</p> Signup and view all the answers

How can the pore size of polyacrylamide gels be adjusted?

<p>Via the concentration of the acrylamide monomer and the ratio of the crosslinking agent, N,N'-methylenebisacrylamide</p> Signup and view all the answers

What type of molecules are typically electrophoresed using polyacrylamide gels?

<p>Proteins and relatively small nucleic acids</p> Signup and view all the answers

How is the agarose gel formed?

<p>Via non-covalent interactions between long polysaccharide chains</p> Signup and view all the answers

How can the pore size of agarose gels be controlled?

<p>Via the concentration of the agarose solution</p> Signup and view all the answers

What happens to the antibodies in a traditional ELISA test upon addition and washing?

<p>They remain free upon addition and are washed off during washing.</p> Signup and view all the answers

What is the purpose of the Scavenger container in Reverse ELISA?

<p>To bind excess antibodies that did not bind to the primary antigens.</p> Signup and view all the answers

What is the advantage of Reverse ELISA over traditional ELISA?

<p>It allows multiple antigens to be tagged and counted at the same time.</p> Signup and view all the answers

What device is used to detect the tagged and bound antibodies in Reverse ELISA?

<p>A detector, such as a flow cytometer.</p> Signup and view all the answers

What is the significance of the sufficient incubation period in Reverse ELISA?

<p>It allows the antibodies to bind to the antigens.</p> Signup and view all the answers

What is the role of enzymatic markers in ELISA assays?

<p>They allow the results of the assay to be measured upon completion.</p> Signup and view all the answers

In what type of setting is Reverse ELISA commonly used?

<p>In the field.</p> Signup and view all the answers

What is the difference between traditional ELISA and Reverse ELISA in terms of test format?

<p>Traditional ELISA uses microtiter plates, while Reverse ELISA does not.</p> Signup and view all the answers

Why is it necessary to increase the concentration of magnesium with high concentrations of dNTPs?

<p>Due to stoichiometric interactions between magnesium and dNTPs that reduce the amount of free magnesium in the reaction medium.</p> Signup and view all the answers

What are the two essential roles of dNTPs in DNA synthesis?

<p>Providing energy and nucleotides needed for DNA synthesis</p> Signup and view all the answers

What is the typical final concentration of dNTPs in a PCR reaction?

<p>About 200 μM</p> Signup and view all the answers

What is the relationship between the number of cycles and the abundance of DNA matrix in PCR?

<p>The number of cycles is inversely proportional to the abundance of DNA matrix.</p> Signup and view all the answers

What is the purpose of ethidium bromide staining in PCR product detection?

<p>To reveal the DNA fragments.</p> Signup and view all the answers

What is the range of primer concentrations used in a PCR reaction?

<p>10-50 pmol per sample</p> Signup and view all the answers

What is the maximum quantity of template DNA that can be used in a PCR reaction?

<p>2 μg</p> Signup and view all the answers

What type of electrophoresis is used in automated systems for PCR product detection?

<p>Capillary electrophoresis.</p> Signup and view all the answers

What is the typical range of Taq polymerase units used per sample in a PCR reaction?

<p>1-3 units</p> Signup and view all the answers

What is required for PCR products to be detected by a laser diode in automated systems?

<p>Primers coupled to fluorochromes.</p> Signup and view all the answers

How does the duration of the temperature cycles depend on the size of the sequence of interest?

<p>The duration should be reduced to a minimum to prevent non-specific amplification, and the elongation time increases with the size of the sequence of interest (1 minute per kilobase).</p> Signup and view all the answers

What is the dynamic range of quantification in real-time PCR assays?

<p>7-8 logarithmic decades.</p> Signup and view all the answers

What is the significance of real-time PCR technology in molecular diagnostics?

<p>It has revolutionized the field, enabling high-throughput, automated technology with lower turnaround times.</p> Signup and view all the answers

Why is it important to minimize the duration of the temperature cycles?

<p>To prevent non-specific amplification</p> Signup and view all the answers

What type of DNA fragments are often visible close to the migration front in agarose gel electrophoresis?

<p>Primer dimers and sometimes primers themselves.</p> Signup and view all the answers

What is the result of nonspecific DNA fragments being amplified in PCR?

<p>Net bands or 'smears'.</p> Signup and view all the answers

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:
    1. Incubate unlabeled antibody with its antigen (sample)
    2. Allow antibodies to bind to antigens
    3. Pass the sample through a Scavenger container with "Scavenger Antigens" bound to its surface
    4. Allow Scavenger Antigens to bind to excess antibodies
    5. 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:
    1. Template DNA: extract from any organism or complex biological material
    2. Primer concentration: 10-50 pmol per sample
    3. dNTPs (deoxyribonucleoside triphosphates): provide energy and nucleotides for DNA synthesis
    4. Matrix DNA: from any organism, not too degraded, and not contaminated with inhibitors
    5. Taq polymerase: 1-3 units per sample
    6. 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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Description

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.

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