Вы находитесь на странице: 1из 15
Transforming education education transforming transforming India Transforming India TERM PAPER : Immunology and Immunotechnology Topic:ELISA(Enzyme Linked
Transforming education education transforming transforming India Transforming India
Transforming education
education transforming
transforming India
Transforming
India

TERM PAPER: Immunology and Immunotechnology

Topic:ELISA(Enzyme Linked ImmunoSorbent Assay)

Transforming education education transforming transforming India Transforming India TERM PAPER : Immunology and Immunotechnology Topic:ELISA(Enzyme Linked

SUBMITTED SUBMITTED TO:-

BY:-

Name: -Nitish Pathania Sarabjot Kaur Sec: -K7802-A06 Reg. No. : -10803694

ACKNOWLEDGEMENT

Mrs.

As The Saying goes “No Work Is Done Without Cooperation”

  • I am extremely grateful and remain indebted to my friends and my guide Mrs. Sarabjot

Kaur for being a source of inspiration and for their constant support in the Design, Implementation and Evaluation of this Term Paper. I am thankful to them for their constant constructive criticism and invaluable suggestions, which benefited me a lot while developing this paper on topic “ELISA(Enzyme Linked ImmunoSorbent Assay)”. Also they provide me a constant source of inspiration and motivation for doing hard work while preparing this term paper. Through this column, it would be my utmost pleasure to express my warm thanks to them for their encouragement, co-operation and consent

without which I mightn’t be able to accomplish this work of Term Paper.

  • I also want to express my gratitude to my God and my parents those are a great source for

me of inspiration. I am again very thankful to Mrs. Sarabjot Kaur who gave me this chance to express my thoughts with the help of this Term paper to make my concepts very clear and make me aware about the immunotechnology of ELISA and its various applications in immunology

Thanking You

Nitish Pathania

Abstract:

ELISAs (Enzyme-Linked-Immuno-Sorbent Assays) have resulted in a revolution in diagnostic procedures in medicine, biochemistry, drug discovery, etc. Enzyme-linked antibodies are used routinely to detect very small amounts of viruses, hormones, and other antibodies! A variant of the ELISA procedure is even used these days by the layman in home pregnancy tests.

The ELISA technique is based on enzyme-linked antibodies, that is an antibody (which is a protein) covalently attached to an enzyme (another protein). In vivo, antibodies (also known as immunoglobulins) are proteins that specifically bind to molecules, which are referred to as antigens that are foreign to the host organism. The antigen may be almost any small molecule or it may be a polypeptide or a portion of a protein or even a carbohydrate. Formation of the antibody-antigen complex is a key step in the immune response and serves to cause elimination of small molecules, or viruses, or even whole cells that are foreign to the host organism. Antigens bind non-covalently and highly specifically to antibodies.

The ELISA procedure takes advantage of the specificity and high affinity of antibodies for a particular antigen to detect minute quantities of a specific antigen in a complex mixture. The detection of a specific antibody-antigen complex relies on measuring the activity of an enzyme covalently linked to the antibody. The enzyme used is usually one that catalyzes a reaction that produces a colored product. Since the enzyme catalyzes the turnover of thousands of substrate molecules, and because absorbance of the dye is proportional to the amount of antibody-antigen complex present in the sample, the ELISA procedure is capable of detecting concentrations of antigen as low as (10 -15 M). Antibodies are produced in vivo by B-cells, which are one of the major classes of cells responsible for the immune response. Antibodies can be produced by injecting an animal (e.g., a goat or a rabbit) with an antigen of interest. Within about one week, the immune system of the animal launches an immune response and many copies of antibody proteins which bind to the antigen are produced. The antibodies can be identified, purified and covalently bound to an enzyme. The enzyme- linked antibody can now act as a specific probe to detect the presence of the antigen in any sample. Many antibodies used in the ELISA procedure are commercially available as enzyme- linked antibodies (also referred to as enzyme linked antibody conjugates). A different enzyme linked antibody would be required, however, for each antigen to be detected.

Why known as

......

?

Enzyme Linked Immunosorbent Assay(ELISA)

1. Antigen of interest is absorbed on to plastic surface (sorbent).

  • 2 Antigen is recognized by specific antibody (immuno). This antibody is recognized by second antibody (immuno) which has enzyme attached

3.

(enzyme-linked).

  • 4. Substrate reacts with enzyme to produce product, usually coloured.

Introduction:

Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. ELISAs are typically performed in 96-well polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from nonbound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It also can be linked to a protein such as streptavidin if the primary antibody is biotin labeled. The most commonly used enzyme labels horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase.

This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substrate.

BASIC PRINCIPLE OF ELISA:

Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).

The enzyme converts a colorless substrate (chromogen) to a colored product, indicating

the presence of Ag: Ab binding. An ELISA can be used to detect either the presence of Antigens or antibodies in a

sample depending how the test is designed. ELISA was dveloped in 1970 and became rapidly accepted

ELISA Formats:

ELISAs can be performed with a number of modifications to the basic procedure. The key step, immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then detected either directly (labeled primary antibody) or indirectly (labeled secondary antibody). The most powerful ELISA assay format is the sandwich assay. This type of capture assay is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies – the capture antibody and the detection antibody.

An ELISA can also be performed as a competitive assay. This is common when the antigen is small and has only one epitope, or antibody binding site. One variation of this method consists of labeling purified antigen instead of the antibody. Unlabeled antigen from samples and the labeled antigen compete for binding to the capture antibody. A decrease in signal from purified antigen indicates the presence of the antigen in samples when compared to assay wells with labeled antigen alone. Fluorescent tags and other alternatives to enzyme-based detection can be used for plate-based assays.

Direct vs. Indirect Detection ELISA Formats:

The direct detection method uses a labeled primary antibody that reacts directly with the antigen. Direct detection can be performed with antigen that is directly immobilized on the assay plate or with the capture assay format. Direct detection is not widely used in ELISA but is quite common for staining of tissues and cells.

The indirect detection method uses a labeled secondary antibody for detection and is the most popular format for ELISA. The secondary antibody has specificity for and the primary antibody. In a sandwich ELISA, it is critical that the secondary antibody be specific for the detection primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen.

Selecting and Coating ELISA Plates:

When developing a new ELISA for a specific antigen, the first step is to optimize the plate- coating conditions for the antigen or capture antibody. Begin by choosing an assay microplate with a minimum protein-binding capacity of 400 ng/cm². The choice of plate color depends upon the signal being detected. Clear polystyrene flat bottom plates are used for colorimetric signals while black or white opaque plates are used for fluorescent and chemiluminescent signals.

Plate coating is achieved through passive adsorption of the protein to the plastic of the assay microplate. This process occurs though hydrophobic interactions between the plastic and non- polar protein residues. Although individual proteins may require specific conditions or pretreatment for optimal binding, the most common method for coating plates involves adding a 2-10 μg/ml solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline (pH 7.4) or carbonate-bicarbonate buffer (pH 9.4). The plate is left to incubate for several hours to overnight at 4-37°C. Typically, after removing the coating solution, blocking buffer is added to ensure that all remaining available binding surfaces of the plastic well are covered. Coated plates can be used immediately or dried and stored at 4°C for later use, depending on the stability of the coated protein.

It is important to note that optimal coating conditions can vary with each protein. With the exception of competition ELISAs, the plates are coated with more capture protein than can actually be bound during the assay in order to facilitate the largest working range of detection possible.

Pre-coated ELISA Plates:

For antibodies and proteins, coating plates by passive adsorption usually works well. However, problems can arise from passive adsorption, including improper orientation, denaturation, poor immobilization efficiency and binding of contaminants along with the target molecule. Antibodies can be attached to a microplate through the Fc region using Protein A, G, or A/G coated plates, which orients them properly and preserves their antigen binding capability. Fusion proteins can be attached to a microplate in the proper orientation using glutathione, metal-chelate, or capture-antibody coated plates.

Antibodies and Probes for ELISA:

Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonals have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the detecting antibody in the sandwich assay to provide improved specificity.

An important consideration in designing a sandwich ELISA is that the capture and detection antibodies must recognize two different non-overlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered.

Another design consideration in choosing antibodies is cost. A polyclonal antibody is generally less expensive (~5 fold) to produce than a monoclonal. The specificity gained by using monoclonals for both the capture and detecting antibody must be weighed against the cost and time required for producing two monoclonal antibodies.

Blocking Buffers and Wash Buffers:

The binding capacity of microplate wells is typically higher than the amount of protein coated in each well. The remaining surface area must be blocked to prevent antibodies or other proteins from adsorbing to the plate during subsequent steps. A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background signal and improving the signal-to-noise ratio.

In addition to blocking, it is essential to perform thorough washes between each step of the ELISA. Washing steps are necessary to remove non-bound reagents and decrease background, thereby increasing the signal : noise ratio. Insufficient washing will allow high background, while excessive washing might result in decreased sensitivity caused by elution of the antibody and/or antigen from the well. Washing is performed in a physiologic buffer such as Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) without any additives

Detection Strategies for ELISA:

The final stage in all ELISA systems is a detection step. Unless a radioactive or fluorescent tag was used, this involves the introduction of an enzyme substrate. The enzyme converts the substrate to a detectable product. If an ELISA has been constructed and developed properly, then the intensity of signal produced when the substrate is added will be directly proportional to the amount of antigen captured in the plate and bound by the detection reagents.

Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic ELISA substrates allow direct visualization and enable kinetic studies to be performed. Fluorescent ELISA substrates are not as common and require a fluorometer that produces the correct excitation beam to cause signal emission to be generated from the fluorescent tag.

ELISA Kits, ELISPOT and In-Cell ELISA:

In addition to the individual components and general principles of ELISA discussed in this article, complete kits are available for detection of specific cytokines and other targets, such as interferon gamma (IFN gamma) and interleukin. ELISA development kits (Mini Kits and Screening Sets) for specific targets include only matched pairs of antibodies and a protocol for coating plates.

ELISPOT kits (enzyme-linked immunospot assay) for measurement of cytokines in single cells are available for human IFN gamma, IL-2 and TNF alpha. An alternative ELISPOT for application to nearly any specific analyte for which antibodies exist is the In-Cell ELISA, developed for performing ELISA assays on plated cells.

ELISA and its Various Types:

The Enzyme-Linked Immuno Sorbent Assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample using two antibodies. One antibody is specific to the antigen and the other reacts to antigen-antibody complexes, and is coupled to an enzyme. This second antibody, which accounts for "enzyme-linked" in the assay's name, can also cause a chromogenic or fluorogenic substrate to produce a signal.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the

enzyme molecules will produce many signal molecules. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations and also for detecting the presence of antigen.

  • 1. Direct ELISA

  • 2. Indirect ELISA

  • 3. Sandwich ELISA

  • 4. Competitive ELISA

  • 5. Multiplex ELISA

2. Indirect ELISA 3. Sandwich ELISA 4. Competitive ELISA 5. Multiplex ELISA Figure 1. Types of

Figure 1. Types of ELISA.

Shown in diagram generalizing models of different types of ELISA. Further variation of the assay are there depending upon the labeling and signal detection methodology. The basic approaches stay the same: fixing either antigen or antibody and detecting antibody-antigen complex.

  • 1. Direct ELISA:

The direct ELISA uses the method of directly labeling the antibody itself. Micro well plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample. However, the direct ELISA requires the labeling of every antibody to be used, which can be a time-consuming and expensive proposition. In addition, certain antibodies may be unsuitable for direct labeling. Direct methods also lack the additional signal amplification that can be achieved with the use of a secondary antibody.

  • 2. Indirect ELISA:

The indirect, two-step method uses a labeled secondary antibody for detection. First, a primary antibody is incubated with the antigen. This is followed by incubation with a labeled secondary antibody that recognizes the primary antibody. For ELISA it is important that the antibody enzyme conjugate is of high specific activity. This is achieved when the antibody is affinity purified and the enzyme conjugation chemistry preserves antibody specificity as well as enzyme activity.

Shown in diagram generalizing models of different types of ELISA. Further variation of the assay are

Table 2. Comparison of Direct and Indirect ELISA Detection Methods

Direct Detection

Advantages of Direct

Quick methodology since only one antibody is used.

Detection

Cross-reactivity of secondary antibody is eliminated.

Disadvantages Direct Detection

of

Immunoreactivity of the primary antibody may be reduced as a result of labeling.

 

Labeling of every primary antibody is time-consuming and expensive.

No flexibility in choice of primary antibody label from one experiment to another.

 

Little signal amplification.

 

Indirect Detection

Advantages of Indirect

A

wide

variety

of

labeled

secondary

antibodies

are

available

Detection

commercially.

 

Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.

Immunoreactivity of the primary antibody is not affected by labeling.

Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.

Different visualization markers can be used with the same primary antibody.

Disadvantages Indirect Detection

of

Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.

 

An extra incubation step is required in the procedure.

 
  • 3. Sandwich ELISA:

The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to the antibody, since at least two antibodies act in the sandwich. For this reason, sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs for quantitation of antigens are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein.

To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate.

Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific.

However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding.

The sensitivity of the sandwich ELISA is dependent on four factors:

The number of molecules of the first antibody that are bound to the solid phase.

The avidity of the first antibody for the antigen.

The avidity of the second antibody for the antigen.

The specific activity of the second antibody.

4. Competitive ELISA: When two “matched pair” antibodies are not available for a target, another option
  • 4. Competitive ELISA:

When two “matched pair” antibodies are not available for a target, another option is the competitive ELISA. The advantage to the competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the antigen or the primary antibody. The example shown in Figure 1 is a labeled antigen as the competitor. In this type of ELISA, there is an inverse relationship between the signal obtained and the concentration of the analyte in the sample, due to the competition between the free analyte and the ligand-enzyme conjugate for the antibody coating the microplate, i.e. the more analyte the lower the signal.

4. Competitive ELISA: When two “matched pair” antibodies are not available for a target, another option
  • 5. Multiplex ELISA:

A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. There are different types of multiplex ELISA have been developed and in practice. One of the examples is to measure antigens by coating or printing capture antibodies in an array format within a single well to allow for the construction of "sandwich" ELISA quantification assays. Generally, multiplex ELISA can also be achieved through antibody array, where different primary antibodies can printed on glass plate to capture corresponding antigens in a biological sample such as plasma, cell lysate, or tissue extract. Detection method can be direct or indirect, sandwich or competitive, labeling or non-labeling, depending upon antibody array technologies.

5. Multiplex ELISA: A logical progression of the widely used microtiter plate ELISA is toward a

ELISA: pros and cons:

Pros:

 

No radioactivity needed: safe and inexpensive

Less expensive equipment

Huge capacity through standard 96-microwell, pattern: miniaturization and

automation. Equal or even better sensitivity than RIA

Versatility: same principle allows for use of different labels, incl. enzymes

Cons:

(coloured end product or light) and fluorochromes.

 

Somewhat more difficult to develop, control and standardize than RIA.

Sigma ELISA (Enzyme Linked Immunosorbent Assay):

An ELISA or enzyme linked immunosorbent assay is a laboratory technique used to detect the presence of antibody in a sample. Medical professionals can use an ELISA test to determine the degree to which a person is allergic to a given antigen.

The first step of an ELISA protocol calls for an ELISA antigen to bind to the inside of a well (96 well plates are commonly used).

A primary antibody is then incubated in the plate. The plate is washed with a buffer before it is incubated with a secondary antibody conjugated to an enzyme. This enzyme will induce a color change in the solution when the appropriate substrate is added. The strength of this color change is measured in a microplate reader and compared to a control sample. The research can then determine how much antibody is present in their sample

When the body's immune system encounters a particular antigen (for example, a characteristic protein on the surface of a virus or bacterium), antibodies that are specific for that antigen intercept it, physically binding to it in a "lock and key"-like fashion, thereby neutralizing the organism.

Enzyme-Linked Immunosorbent Assay (ELISA) utilizes an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biological activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye.

Sigma ELISAs Protocol:

  • 1. A monoclonal or polyclonal antibody (capture antibody) specific for protein is coated into the wells of a multi well plate (96 wells in a plate).

  • 2. Antigen in standards, unknown samples and controls is added and incubated in order to

facilitate binding of antigen and antibody.

  • 3. Several washes are performed after each incubation in order to remove excess of unbound

reagents.

  • 4. A second (detection) antibody specific for the whole protein at specific site is added and

incubated 1 hour at RT.

  • 5. During the second incubation, this antibody serves as a detection antibody by binding to

another epitope of the immobilized antibody.

  • 6. After removal of excess detection antibody, HRP-IgG is added and binds to the detection

antibody to complete the four-membrane sandwich.

  • 7. Substrate solution is added, which is acted upon by the bound enzyme to produce color.

The intensity of this colored product is directly proportional to the concentration of antigen

present in the original sample.

  • 8. The optical density measured at 450 nm in the multiwell plate reader is used to calculate

the concentration of protein in question.

Applications:

Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test or West Nile Virus).

It

has

also

found

applications

in

the food industry

in

detecting

potential food

 

ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of

drugs. Enzyme-Linked Immunosorbent Assays (ELISA) is widely used in research, diagnosis, and testing because of they are affordable and sensitive to tiny amounts of material.

Usually,

the

assay

involves

multiple binding and rinsing steps, followed by

spectrophotometry. However, in some cases, properly engineered assays can be done in a single step giving qualitative results readable by eye. The pregnancy tests commonly available in drug stores use ELISA to detect the human

chorionic gonadotrophin hormone in urine.

ELISA test for HIV detection:

The enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.

In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to human antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.

References:

http://www.piercenet.com/Proteomics/browse.cfm?fldID=F88ADEC9-1B43-4585-922E-836FE09D8403

http://en.wikipedia.org/wiki/ELISA

www.biosupply.co.uk/doc.php?id=2601

http://www.bio.umass.edu/micro/immunology/elisa/elisa0.htm

http://thefutureofthings.com/articles/37/biopen-senses-biothreats.html

http://www.genwaybio.com/gw_file.php?fid=6056

G.L. Anderson and L. A. McNellis, J. Chem. Educ., 75, 1275 (1998)