My ELISA seminar

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ELISA is an important analytical technique for estimation and quantification of protein in a complex mixture.

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ELISA: 

ELISA Somsubhra Pal I M.Pharm Pharmacology

Enzyme Linked immuno sorbent Assay: 

Enzyme Linked immuno sorbent Assay Presented By: Somsubhra Pal I M.Pharm Pharmacology Facilitated By: Mr. Mukund Handral Asst. Professor PESCP

What is an ELISA?: 

What is an ELISA? The enzyme linked immunosorbent assay (ELISA) is a powerful method for detecting and quantifying a specific protein in a complex mixture. This method enables analysis of protein samples immobilized in microplate wells using specific antibodies. This method was originally described by Engvall and Perlmann in 1971.

The basic elements of ELISA: 

The basic elements of ELISA Coating/Capture: direct or indirect immobilization of antigens to the surface of polystyrene microplate wells. Plate Blocking: addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.

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3) Probing/Detection : incubation with antigen-specific antibodies that affinity-bind to the antigens. 4) Signal Measurement: detection of the signal generated via the direct or secondary tag on the specific antibody.

Different types of ELISA: 

Different types of ELISA Direct ELISA Indirect ELISA Competition ELISA Sandwich ELISA

Direct ELISA: 

Direct ELISA The antigen is immobilized to the wells of the microplate. The plate is blocked and the antigen is probed with a specific detection antibody. The detection antibody is directly labelled with an enzyme which is bound by an enzyme substrate. The signal observed is proportional to the antigen in the sample.

Indirect ELISA: 

Indirect ELISA Indirect ELISA is different from Direct ELISA in that the primary antibody is probed with an enzyme or fluor-labelled secondary antibody. This enzyme is bound to by the enzyme substrate and a signal is generated.

Competition ELISA: 

Competition ELISA In a competition ELISA, the assay is based on competition between the antigen in a standard/sample and an enzyme conjugated version of the same antigen for a limited amount of antibody bound to a pre-coated plate. Both are mixed together in the same well.

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As the concentration of antigen in the sample increases, the amount of labeled antigen captured by the coating antibody decreases. Therefore there is an inverse relationship between optical density (OD) and the amount of analyte in the sample.

Sandwich ELISA: 

Sandwich ELISA In Sandwich ELISA the first antibody, called Capture Antibody or Coated Antibody is bound to the plate. The antigen is then immobilized. The second antibody, called the Detection antibody detects the antigen. A third antibody housing the enzyme is added which gives a signal on addition of substrate.

Concepts of ELISA: 

Concepts of ELISA Combination of monoclonal and polyclonal antibodies can be used in Sandwich ELISA. Monoclonal antibodies are used as Coating and Polyclonal antibodies as Detection antibodies. Antigen Immobilization: Antigen immobilization to the plate can occur by passive adsorption, usually using a 0.2M carbonate/bicarbonate buffer at pH>9.

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But when antigen is present in very low concentration or does not adhere well to the plates, then the alternative sandwich ELISA is employed. In Sandwich ELISA only specific antigen becomes mobilized. Hence it has gained immense popularity because specific protein can be analyzed from complex protein samples.

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Direct ELISA Indirect ELISA No secondary antibody is used in Direct ELISA. The primary antibody is labelled with enzyme. Secondary Antibody is used. It is labelled with enzyme. Direct ELISA is faster and potential background signal from secondary antibody cross-reactivity with the coating antibody is also eliminated. Indirect ELISA is much slower. Background signal exists. Direct ELISA is less sensitive and best used when target is abundant. Indirect ELISA is much more sensitive.

Fluorescence: 

Fluorescence Fluorophores can be used to generate a fluorescent signal. This approach is common in Multiplex arrays, as more than one antigen can be detected simultaneously with antibodies conjugated to different fluorophores. The detection limit is around 100pg/well which is less sensitive than Colorimetric or Chemiluminescent detection.

Enzymes used in ELISA: 

Enzymes used in ELISA Two enzymes are commonly used in ELISA. Alkaline Phosphatase(AP) is used in a minority of assays. Its size(140 Kda) makes it difficult to conjugate more than one or two molecules of the enzyme to each molecule of an antibody, thus only limited amount of signal can be generated.

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To the contrary Horseradish Peroxidase(HRP) has a small size of 40 KDa and can allow more molecules to be coupled to antibodies. SUBSTRATES Enzymatic signal generation requires the catalysis of a substrate to produce a colored or fluorescent compound or chemiluminescence (visible light). Colorimetric substrates produce a colored product that accumulates over time.

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It is relative to the amount of enzyme present in each well. In Chemifluorescent detection the generated product is fluorescent. The signal can be estimated with the help of a Fluorometer. Chemiluminescnece generates energy released in the form of light. In Chemiluminescence the most common approach is to use Luminol in the presence of HRP and Peroxide buffer.

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Luminol is oxidized and forms an excited state product that emits light as it decays to ground state. Types of plates Plates used in conventional ELISA applications are typically made of polystyrene. Other materials such as polypropylene, polycarbonate and in some instances nylon are occasionally used.

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Plates can be gamma-irradiated to impart a positive charge, which aids the coating procedure. Fluorescent detection requires the use of an opaque black plate as the background is lower. Chemiluminescent detection demands the use of white plates to magnify the signal.

Antibodies: 

Antibodies For an antibody to work in ELISA it must react specifically with antigen. It must not cross-react with the blocking buffer. In Sandwich type ELISA it is essential that the two different type of antibodies react with different epitopes on the antigen. The concentration of the antibodies should be optimized.

Blocking Buffer: 

Blocking Buffer The purpose of Blocking Buffers is to prevent non-specific binding of proteins to the plate. Surfactants like Tween-20 can be added to the blocking solution. It minimized hydrophobic interactions between the blocking proteins and the antigens and the antibodies.

Target Antigen and Plate pre-coating: 

Target Antigen and Plate pre-coating The 3D structure of an antigen may be altered during adsorption to the plate. In this case, the plate can be pre-coated with a binding protein(such as capture antibody).

Washing of Plate: 

Washing of Plate Tris -buffered saline and Phosphate buffered saline containing 0.05% v/v Tween-20 are wash buffers used to wash plates. Generally at least 3 x 5 minute washes should be applied after the incubation of coating antibody, sample and detection antibody. 6 x 5 minute washes should be given after incubation with the enzyme conjugate.

Signal Detection: 

Signal Detection Colorimetric substrates are measured using a standard plate reader with the appropriate filters. Chemifluorescence is measured using a fluorometer with the appropriate excitation and emission filters. Chemiluminescence is most commonly measured using a luminometer.

Applications of ELISA: 

Applications of ELISA ELISA can be used to detect potential food allergens, such as milk, peanuts, walnuts, almonds etc. It is a employed as a screening test for HIV. It can be used in Toxicology to screen drugs.

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The other uses of ELISA include : detection of Mycobacterium antibodies in tuberculosis. detection of rotavirus in feces . detection of hepatitis B markers in serum. detection of enterotoxin of E. coli in feces . detection of HIV antibodies in blood samples.

References: 

References Steimer W, Przyklenk B, Bauernfeind A. Processing raw absorbance data generated by ELISA for measuring specific antibody activity. Serodiagnosis and Immunotherapy in Infectious Disease. 1993;5(1):53-9. Gut- Winiarska M, Jacobs L, Kerstens H, Bienkowska-Szewczyk K. A highly specific and sensitive sandwich blocking ELISA based on baculovirus expressed pseudorabies virus glycoprotein B. Journal of Virological Methods. 2000;88(1):63-71. URL: http://www.piercenet.com/browse.cfm?fldID=EE79C527-5056-8A76-4E92-2E2C1E1643AB