ELISA Assay Applications

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?

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

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

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

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

Coeliac disease. Signup and view all the answers

What is the purpose of ELISA in toxicology?

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

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

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

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

Dengue. Signup and view all the answers

What are the two dominant compounds used for gel electrophoresis?

Polyacrylamide and agarose Signup and view all the answers

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

Alkaline phosphatase. Signup and view all the answers

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

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

How can the pore size of polyacrylamide gels be adjusted?

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

What type of molecules are typically electrophoresed using polyacrylamide gels?

Proteins and relatively small nucleic acids Signup and view all the answers

How is the agarose gel formed?

Via non-covalent interactions between long polysaccharide chains Signup and view all the answers

How can the pore size of agarose gels be controlled?

Via the concentration of the agarose solution Signup and view all the answers

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

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

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

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

What is the advantage of Reverse ELISA over traditional ELISA?

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

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

A detector, such as a flow cytometer. Signup and view all the answers

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

It allows the antibodies to bind to the antigens. Signup and view all the answers

What is the role of enzymatic markers in ELISA assays?

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

In what type of setting is Reverse ELISA commonly used?

In the field. Signup and view all the answers

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

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

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

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

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

Providing energy and nucleotides needed for DNA synthesis Signup and view all the answers

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

About 200 μM Signup and view all the answers

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

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

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

To reveal the DNA fragments. Signup and view all the answers

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

10-50 pmol per sample Signup and view all the answers

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

2 μg Signup and view all the answers

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

Capillary electrophoresis. Signup and view all the answers

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

1-3 units Signup and view all the answers

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

Primers coupled to fluorochromes. Signup and view all the answers

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

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). Signup and view all the answers

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

7-8 logarithmic decades. Signup and view all the answers

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

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

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

To prevent non-specific amplification Signup and view all the answers

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

Primer dimers and sometimes primers themselves. Signup and view all the answers

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

Net bands or 'smears'. 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.