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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Biotin Interference on Diagnostic Testing

biotin-b7

Biotin, also known as vitamin B7 is a coenzyme that is involved in carbon dioxide transfer in carboxylase reactions. The USDA recommended dietary reference intake for biotin is 30 ug per day which should mostly come from food. The last few years biotin has been marketed heavily as a beauty supplement. It is used in hair, skin, and nail supplements, and is not FDA regulated and is sold as over-the-counter. Biotin can be found in B-complex vitamins, multivitamins, prenatal vitamins, vitamin H, and vitamin B7 supplements. The only FDA recommended use for biotin is in patients with secondary progressive multiple sclerosis who receive mega-doses of up to 300 mg per day. Even in such large doses biotin is considered nontoxic and has very little adverse effects.

The issue is that serum or plasma biotin may potentially interfere with any assay that uses biotin-streptavidin binding. Biotin is a small molecule that attaches covalently to a variety of targets with minimal effect on their biological activity. The biotin binding makes the target an easy capture because it forms a strong bond with avidin, streptavidin, and NeutrAvidin proteins who have an exceptionally high affinity for biotin. Biotin-streptavidin detection is a favorite among many immunoassays across many manufacturers including Roche, Ortho, Beckman, Siemens, and Dimension.

The direction of interference depends on the design of the assay. Some results may be falsely elevated, and some may be falsely decreased. The sandwich and competitive assays are among the most commonly impacted. Interference can occur with hormone tests such as parathyroid hormone (PTH), thyroid stimulating hormone (TSH), T4, T3, and even troponin tests.

Sandwich assays involve two antibodies that form a sandwich with the analyte being tested to be measured. The first antibody is labeled with a signal that can be quantified and the other antibody to the target is labeled with biotin. When the biotin:antibody complex binds to streptavidin-coated beads, the labeled antibody then binds creating a sandwich. The resulting complex is then measured. The more complexes that are created, the stronger the signal, i.e the more target analyte there is. Excess free biotin interferes by binding to the streptavidin-coated beads, leaving fewer binding opportunities for the antibodies. Antibody complexes that have successfully bound the analyze get washed away and are then undetected, resulting in falsely low results.

Competitive assays consist of an antibody to the analyte that is labeled with biotin. The analyte must compete for antibody binding sites with a reagent that is a supplied version of itself with a label for detection. If no analyte is present, the reagent occupies all the antibody binding sites and the complex is captured by streptavidin, and a strong signal is emitted. If analyte is present, that occupies antibody binding sites that outcompete the labeled reagent. When analyte is present, there is less detection and less signal measured. It is an inverse relationship. When analyte is not present, there is a strong signal detected, when analyte is present, there is a weak signal detected. Free biotin sticks to the streptavidin, leaving fewer antibody binding sites for the analyte:antibody or reagent:antibody complex. The complexes get washed away and causes weakening of the signal. This may give the impression that analyte is present, even in its absence.

This is an ongoing issue and the FDA advises the healthcare community; patients and physicians both to disclose any supplements that are being taken that contain biotin. Physicians should advise laboratory if interference from biotin is a possibility. Practice should be implemented to counsel patients to abstain from oral biotin 2-3 days before blood tests. Biotin has a rapid half-life of 2 hours, but patients taking mega-doses (>30 mg) have demonstrated interference on laboratory tests for up to 24 hours.

Physicians should educate patients to increase awareness of biotin interference. Adverse health effects can occur if test results are falsely skewed in any direction.