Enzyme-Linked Immunosorbent Assays (ELISA)

The first step in any ELISA assay is the immobilization of the antigen within the sample to the wall of the wells within a microtiter plate. These microtiter plates are usually 96-wells. This is by direct adsorption to the plates surface or by using a capture antibody. The capture antibody has to be specific to the  target antigen. After immobilization, another antibody is added called the detection antibody. This detection antibody binds to the adsorbed antigen which forms an antigen:antibody complex. This detection antibody is either directly conjugated to an enzyme, such as horseradish peroxidase (HRP), or provides an antibody-binding site for a secondary labeled antibody. There are four different types of ELISAs which will all be discussed below. ELISAs take advantage of an enzymatic label to produce a signal that can be quantified and correlated to the binding of an antibody to an antigen. The final assay signal is measured using spectophotometry.

Direct ELISA

In the direct ELISA, the detection antibody is conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP). These substrates produce a colorimetric output that is then measured. The advantages of a direct ELISA is that it is a short protocol which saves time and reagent, and money. There is no cross-reactivity from a secondary antibody that can cause interference. The disadvantages are that there is no signal amplification, so the primary antibody must be conjugated for it to work.

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Indirect ELISA

In the indirect ELISA, antibodies can be conjugated to biotin, which is then followed by a streptavidin-conjugated enzyme step. This is becoming more common place within the clinical laboratory. Alternatively, the detection antibody is typically a human IgG antibody that binds to the antigen within the wells. This primary antibody has multiple antibody-binding sites on it. A secondary rabbit anti-human IgG antibody conjugated with an enzymatic substrate is added. This secondary antibody binds to the first antibody and gives off a colorimetric signal which can be quantified by spectrophotometry. There are advantages over the direct ELISA, mainly that there is signal amplification by using several antibodies, allowing for high flexibility. This also creates a longer protocol, and increases the chances for cross-reactivity, which can be deemed disadvantages.

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Sandwich ELISA

The sandwich ELISA is less common, but is highly efficient in antigen detection. It quantifies antigens using multiple polyclonal or monoclonal antibodies. Monoclonal antibodies recognize a single epitope, while a polyclonal antibody recognizes multiple antigen epitopes. The antigen that is to be measured must contain at least two antigenic epitopes capable of binding to an antibody for this reason. The first step is to coat the microtiter plate wells with the capture antibody within a carbonate/bicarbonate buffer (pH 9.6). Proceed to incubate the plate overnight at 4 degrees Celsius. Wash the plate twice using PBS. Incubate the plate again for at least 2 hours at room temperature. Wash the plate again using PBS. The next step is to add diluted unknown samples to each well. Its important to run unknown samples against those of a standard curve by running standards in duplicates or triplicates. Incubate for 90 minutes at 37 degrees Celsius. then remove the sample and wash with PBS again. Next, add diluted detection antibody to each well. Its important to make sure that the detection antibody recognizes a different epitope on the target antigen than the capture antibody. The prevents interference with antibody binding. To maximize specificity and efficiency, use a tested matched pair. Once the detection antibody has been added, incubate for 2 hours at room temperature. Wash once again with PBS. After washing, add conjugated secondary antibody to each well. Incubate once again at room temperature, then proceed to wash. Once again, horseradish peroxidase and alkaline phosphatase are used as enzymes conjugated to the secondary antibody. The substrates for HRP are called HRP chromogens. Cleavage of hydrogen peroxide is coupled to an oxidation reaction which changes color. Another common substrate used is ABTS. The end product is green.

Sandwich-ELISA

The sandwich ELISA employs high specificity, even when using complex samples. Within the sandwich ELISA, both direct and indirect methods can be used. It can be challenging to find two different antibodies against the same target the recognize different epitopes.

Competitive ELISA

The competitive ELISA is exactly what its name suggests; it is a competitive binding process which is produced by the sample antigen, and an add-in known concentration of antigen. A primary unlabeled antibody is incubated with the unknown sample antigen. This creates antigen:antibody complexes, which are then conjugated to a microtiter plate which is pre-coated with the same antigen. Any free antibody binds to the same antigen on the well. Unbound antibody is removed by washing the microtiter plate. The more antigen within the unknown sample means that less antibody will be able to bind to the antigens within the wells, hence the assay gets its name. Its a competition. A secondary conjugated antibody that is specific for the primary antibody bound to the antigen on the pre-coated on the wells is added. When a substrate is added, the reaction elicits a chromogenic or fluorescent signal. The higher the sample antigen concentration, the weaker the eventual signal.

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References

https://www.bio-rad-antibodies.com/elisa-procedure.html

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html

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The Antibody

An antibody or immunoglobulin is a large Y-shaped protein produced primarily by plasma cells of the humoral immune system. They are used to recognize and neutralize any foreign antigens or pathogens. An antibody is identical to the B-cell receptor of the cell that secretes it except for a small portion of the C-terminus of the heavy-chain constant region. The difference is that a B-cell receptor C-terminus is a hydrophobic membrane-anchoring sequence and on an antibody, the C-terminus is a hydrophilic sequence that allows its secretion. The Y-portion of the consists of two arms that vary between the different antibody molecules, otherwise known as the V-region. The V-region is involved in antigen binding. The C-region is far less variable and is the part of the molecule that interacts with effector cells and other molecules. All antibodies are constructed in the same way paired from heavy and light polypeptide chains joined by disulfide bonds so that each heavy chain is linked to a light chain and the two heavy chains are linked together.

There are two types of light chains, lambda and kappa. A given immunoglobulin has one or the either, never both. In humans the ratio of kappa to lambda; the two types of light chains in immunoglobulins is 2:1. The class, and the effector function of an antibody is defined by the structure of its heavy chain. There are five main heavy-chain isotypes. The five major immunoglobulin classes are IgM, IgD, IgG, IgA, and IgE. IgG is the most abundant immunoglobulin and has several subclasses (1, 2, 3, and 4 in humans). The distinctive functional properties are conferred by the carboxyl -terminal part of the heavy chain, where it is not bonded with the heavy chain.

Each chain of the immunoglobulin consists of a protein domain. Each protein domain consists of a series of similar, but not identical sequences about 110 amino acids long . The light chain is made up of two domains, and the heavy chain consists of four. The variable or V-domain of the heavy and light chains together consist of the V-region of the antibody allowing it to bind specific antigens. The constant domains of the heavy and light chains together make up the C-region. The V-region or the Y of the molecule, where the antigen binding activity takes place is called the Fab fragments. Fab stands for fragment antigen binding. The other part of the molecule, the constant region (C-region) contains no antigen-binding activity, and is called the Fc fragment. Fc stands for Fragment crystallizable. This is the part of the molecule that interacts with effector molecules and cells.

The immunoglobulin molecule is flexible. There is a hinge region that links the Fc and Fab regions of the molecule, allowing independent movement of the two Fab arms.

Recap

To recap. An antibody molecule is made up of four polypeptide chains, comprising of two identical light chains and two identical heavy chains, which can be thought of as forming a flexible Y-shaped structure. Each of the four chains has a variable (V) region at its amino terminus, which contributes to the antigen-binding site, and a constant (C) region, which determines the isotype of the immunoglobulin. The light chains are bound to the heavy chains are non-convalent disulfide bonds. The V-regions of the light and heavy chains pair together to form the Fab region on the arms of the Y-structure. The trunk of the Y-structure, consisting of the carboxyl-terminal domains of the heavy chains make up the Fc fragment. The Fc fragment determines the different isotype of the immunoglobulin and interacts with different effector molecules. There is a hinge region joining the Fab and Fc regions allowing the antibody independent movement to maximize its antigen binding capabilities.

 

 

 

The Precipitation Curve

This article will review basic immunology principles by defining key terms and explaining different techniques and phenomenons.

Key Definitions

Sensitization is the basic reaction of an antigen and an antibody binding. During an antigen:antibody reaction, the antigen or the antibody can be measured using a variety of methods. Each method has its advantages and disadvantages.

These reactions are sensitive and there are multiple external factors that affect the effectiveness of the reaction. The temperature, pH and concentration of the reactants effect the reaction itself. The length of incubation also affects the reaction. This principle applies to doing an indirect antiglobulin test for pre-transfusion testing. The reaction needs to incubate at 37 degrees celsius for a minimum of 15 minutes to properly allow the IgG antibodies to react and form a complex with their specific antigen.

The antigen:antibody reaction has three distinct phases; the primary phenomenon is the initial combination of a single antibody binding to its corresponding single antigen. The secondary phenomenon is where these single antibody:antigen reactions create a lattice formation to create large molecules which are easily detectable. The tertiary phenomenon is the effect that these immune complexes have within the tissues; this could be inflammation, phagocytosis, deposition of the immune complexes, immune adherence, and chemotaxis.

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The primary reaction of an antigen and an antibody depends on two defining characteristics; affinity and avidity. Affinity is the initial force of attraction that an antibody has for its specific antigenic epitope or determinant. Avidity is the sum of all attractive forces between an antigen and an antibody. The stronger the chemical bonds that hold the antibody:antigen complex together, the less likely that the reaction will reverse.

Precipitation involves the combination of a soluble antibody with a soluble antigen which produces insoluble complexes.

Agglutination is the process which particulate antigen aggregate to form visible complexes if the specific antibody is present.

Complement fixation is the triggering of the classical complement pathway due to the combination of the antigen with its specific antibody.

The Precipitin Curve

Precipitation reactions are dependent on the amount of antigen and antibody present in the test system. The precipitin curve is a graphic representation of these reactions that occur when the concentration of one reactant is constant for every test sample, while the concentration of the second reactant is increased serially in the test samples. The two reactants can be interchangeable, so the constant in any given reaction can either be the antigen or the antibody. For the purpose of this article, the antibody is going to be the constant. The addition of low concentrations of antibody allows the formation of soluble immune complexes, however as the concentration of the antigen is increased, precipitation is observed. The precipitin is the insoluble complexes. The antigen concentration continues to rise until the maximum amount of precipitin is reached. This point is called the equivalence point. The equivalence point is where there is optimum proportions of antigen and antibody to result in lattice formations to form insoluble immune complexes. When antigen concentration continues to rise past the equivalence point, the precipitin observed decreases. The curve is classed into three regions.

The early stage of the precipitin curve before the equivalence point is called the prozone and it is a zone of antibody excess. In the zone of antibody excess, there is insufficient antigen to form the large immune complexes comprised of extensive cross-linking. Its because of this principle that there will be false negative reactions. As more antigen is added, these complexes are able to form and it reaches the equivalence point.

The late stage of the precipitin curve is called the postzone and it is the zone of antigen excess. When there is an increasing amount of antigen added beyond the zone of equivalence, there is a gradual decrease in the amount of precipitin observed, until finally there is zero precipitation observed. There is free antigen is the solution. At this point all the antibody binding sites are saturated by multiple antigens and as a result there is less cross-linking leading to soluble immune complexes. This also leads to a false negative reaction.

To recap on what has been learned; There is a precipitation curve that represents the proportion of antigen and antibody concentrations, one being constant, and the other being added in serial additions. The postzone is the zone of antibody excess, resulting in the inability to form cross-linked immune complexes resulting in false negative reactions. The prozone is the zone of antigen excess which also leads to a failure to form cross-linked immune complex. The prozone, just like the postzone, results in a false-negative reaction.