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Can someone simplify Enzyme Linked Immunosorbent Assay (EIA)?

Enzyme-linked Immunosorbent Assay (shortened as ELISA) is used to identify peptides, proteins, antibodies and hormones. Also, called as enzyme immunoassay (EIA), ELISA finds use in the fields of biotechnology and medicine as a diagnostic tool. Mainly, antibodies and color changes are used to identify target substances. Also, ELISAs are useful in measuring antigen and antibody concentration.

Before the advent of ELISA, radioimmunoassay employing radioactively-labelled antigens and antibodies were used. Radioactivity served as the reporter signal indicating specific antigen or antibody. As radioimmunoassay posed significant health risks to researchers, alternatives were sought.

In 1960s, Enzyme linking process was developed by two different teams spearheaded by Stratis Avrameas and G. B. Pierce. In the same period, immunosorbent preparation technique was published by Wide and Jerker Porath. Independent research papers published in 1971 by Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands produced the knowledge that go into making ELISA.

The conventional ELISA involves usage of chromogenic reporters and substrates to produce color changes to indicate the presence of specific antigen or an analyte. Newer Assay techniques make use of fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. The advantage of using advanced reporters help in measuring multiple analytes in a single or cycle of assays (Multiplexing) and higher sensitivities (specificity and sensitivity)

Technically, newer assays use reporters that are not enzymes in most cases, nonetheless the underlying principles of the assays are similar. Therefore, these assays are grouped as ELISAs.

ELISA works by coupling antibody or antigen to assay enzyme. The assay combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens. The sensitivity and precision of the assay is enhanced by coating the plate with high-affinity antibodies.

To know more about ELISA Types, advantages and disadvantages and methods, refer ELISA page in Mybiosource Learning Center.

What are the advantages and limitations of using Western-blot vs. ELISA vs. flow cytometry?

First, let us discuss the definition of Western blotting, ELISA and Flow cytometry in simple language. You can always follow the link to get more detailed information on these assays/methods.

ELISA or Enzyme-linked Immunosorbent Assay is useful in identifying a substance using antibodies and color changes.

Western blotting is used to separate and identify proteins. Through gel electrophoresis, the proteins are separated based on molecular weight. Find protocol and detailed explanation on western blotting in mybiosource learning Center.

Flow cytometry is useful in analyzing physical and chemical properties of particles. In biological research, cell components are fluorescently labelled and then excited by a laser beam to emit light at different wavelengths. Relative granularity, size, fluorescence intensity and internal complexity is mainly studied via flow cytometry. Find protocol and detailed explanation on flow cytometry in mybiosource learning Center.

How are antibodies detected?

 Initially, identification and characterization of antibodies were limited to the field of scientific research. As the measurement and identification of bacterial infections, viral infections, autoimmune diseases and allergies became more and more important in clinical settings, methods of detecting antibodies have evolved big time and newer/efficient methods are being offered.

Two important methods for detection of antibodies actively used are immunoprecipitation assay and immunoblotting. While there are assays that are superior to immunoblotting, the cost factor (specific to clinical settings) plays a role in making those assays economically not viable.

Immunoprecipitation

To analyze a protein, you must have a reliable detection system that unambiguously enables you to follow the target protein. This is especially true when the target molecule is in crude or even semipurified form. The purification of a bioactive molecule is frequently accomplished by using a definitive assay designed to recognize a property of the target protein. You can read and find protocol of Immunoprecipitation in mybiosource learning center.

Immunoblotting

The techniques use antibodies to identify target proteins from sample protein species. It involves identification of target protein via antigen-antibody specificity.  Proteins are separated by electrophoresis and transferred onto membranes. The membrane is overlaid with a specific target (primary antibody) and a secondary labeled antibody i.e. enzymes or radioisotopes. You can read and find protocol of Immunoblotting in mybiosource learning center.

How do direct and indirect ELISA differ?

Direct ELISA: Quicker of all the ELISA, the direct assay is used in detection of antigens coated to the multiwell plate by an antibody conjugated to an enzyme. The assay is less consuming in terms of time, steps and reagents.

Indirect ELISA: The assay is usually carried out in two stages. Like Direct ELISA, the antigen is coated to a polystyrene multiwell plate. In the first stage, unlabeled primary antibody is introduced into the well which is specific to an antigen. In the second stage, enzyme labeled secondary antibody (often polyclonal antibody) is introduced to the well.

The advantages of Indirect ELISA include enhanced sensitivities since more than one labeled antibodies are used for bounding with primary antibody. The experimental procedure can be made flexible as per the demands of the study given the stages in Indirect ELISA.

Can someone simplify Enzyme Linked Immunosorbent Assay (EIA)?

Enzyme-linked Immunosorbent Assay (shortened as ELISA) is used to identify peptides, proteins, antibodies and hormones. Also, called as enzyme immunoassay (EIA), ELISA finds use in the fields of biotechnology and medicine as a diagnostic tool. Mainly, antibodies and color changes are used to identify target substances. Also, ELISAs are useful in measuring antigen and antibody concentration.

Before the advent of ELISA, radioimmunoassay employing radioactively-labelled antigens and antibodies were used. Radioactivity served as the reporter signal indicating specific antigen or antibody. As radioimmunoassay posed significant health risks to researchers, alternatives were sought.

In 1960s, Enzyme linking process was developed by two different teams spearheaded by Stratis Avrameas and G. B. Pierce. In the same period, immunosorbent preparation technique was published by Wide and Jerker Porath. Independent research papers published in 1971 by Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands produced the knowledge that go into making ELISA.

The conventional ELISA involves usage of chromogenic reporters and substrates to produce color changes to indicate the presence of specific antigen or an analyte. Newer Assay techniques make use of fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. The advantage of using advanced reporters help in measuring multiple analytes in a single or cycle of assays (Multiplexing) and higher sensitivities (specificity and sensitivity)

Technically, newer assays use reporters that are not enzymes in most cases, nonetheless the underlying principles of the assays are similar. Therefore, these assays are grouped as ELISAs.

ELISA works by coupling antibody or antigen to assay enzyme. The assay combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens. The sensitivity and precision of the assay is enhanced by coating the plate with high-affinity antibodies.

To know more about ELISA Types, advantages and disadvantages and methods, refer ELISA page in Mybiosource Learning Center.

What are the advantages and limitations of using Western-blot vs. ELISA vs. flow cytometry?

First, let us discuss the definition of Western blotting, ELISA and Flow cytometry in simple language. You can always follow the link to get more detailed information on these assays/methods.

ELISA or Enzyme-linked Immunosorbent Assay is useful in identifying a substance using antibodies and color changes.

Western blotting is used to separate and identify proteins. Through gel electrophoresis, the proteins are separated based on molecular weight. Find protocol and detailed explanation on western blotting in mybiosource learning Center.

Flow cytometry is useful in analyzing physical and chemical properties of particles. In biological research, cell components are fluorescently labelled and then excited by a laser beam to emit light at different wavelengths. Relative granularity, size, fluorescence intensity and internal complexity is mainly studied via flow cytometry. Find protocol and detailed explanation on flow cytometry in mybiosource learning Center.

How are antibodies detected?

 Initially, identification and characterization of antibodies were limited to the field of scientific research. As the measurement and identification of bacterial infections, viral infections, autoimmune diseases and allergies became more and more important in clinical settings, methods of detecting antibodies have evolved big time and newer/efficient methods are being offered.

Two important methods for detection of antibodies actively used are immunoprecipitation assay and immunoblotting. While there are assays that are superior to immunoblotting, the cost factor (specific to clinical settings) plays a role in making those assays economically not viable.

Immunoprecipitation

To analyze a protein, you must have a reliable detection system that unambiguously enables you to follow the target protein. This is especially true when the target molecule is in crude or even semipurified form. The purification of a bioactive molecule is frequently accomplished by using a definitive assay designed to recognize a property of the target protein. You can read and find protocol of Immunoprecipitation in mybiosource learning center.

Immunoblotting

The techniques use antibodies to identify target proteins from sample protein species. It involves identification of target protein via antigen-antibody specificity.  Proteins are separated by electrophoresis and transferred onto membranes. The membrane is overlaid with a specific target (primary antibody) and a secondary labeled antibody i.e. enzymes or radioisotopes. You can read and find protocol of Immunoblotting in mybiosource learning center.

How do direct and indirect ELISA differ?

Direct ELISA: Quicker of all the ELISA, the direct assay is used in detection of antigens coated to the multiwell plate by an antibody conjugated to an enzyme. The assay is less consuming in terms of time, steps and reagents.

Indirect ELISA: The assay is usually carried out in two stages. Like Direct ELISA, the antigen is coated to a polystyrene multiwell plate. In the first stage, unlabeled primary antibody is introduced into the well which is specific to an antigen. In the second stage, enzyme labeled secondary antibody (often polyclonal antibody) is introduced to the well.

The advantages of Indirect ELISA include enhanced sensitivities since more than one labeled antibodies are used for bounding with primary antibody. The experimental procedure can be made flexible as per the demands of the study given the stages in Indirect ELISA.