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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Pheochromocytoma Workup

A pheochromocytoma is a catecholamine-secreting tumor that arises from the chromatin cells of the adrenal medulla. Extraadrenal pheochromocytoma arise from the sympathetic ganglia and are referred to as catecholamine-secreting paragangliomas. These neoplasms are very rare, occurring in less than 0.2% of the patients. Most catecholamine-secreting tumors are sporadic and occur in the fourth to fifth decade of life, effecting both men and women equally. However, about 40% of the patients that present with a pheochromocytoma there is a familial inheritance. These familial tumors arise earlier in life and typically associated with several familial disorders. Such disorders are Von Hippel-Lindau (VHL) syndrome, multiple endocrine neoplasia type 2 (MEN2), and neurofibromatosis (NF1).

Clinical Presentation

Symptoms and signs only occur in about 50% of patients and paroxysmal in nature. There is a classic triad of symptoms that is observed which consists of episodic headache, sweating, and tachycardia with either paroxysmal hypertension or primary hypertension. Paroxysmal hypertension is the most common sign of pheochromocytoma. The headache associated with pheochromocytoma can vary from mild to severe and occurs in 90% of patients. Generalized sweating occurs accompanied by forceful palpitations, tremors, dyspnea, fatigue, and often anxiety and panic attack-type symptoms. There is increased secretion of catecholamines; epinephrine, norepinephrine, and dopamine which cause abnormalities in carbohydrate metabolism which leads to insulin resistance, and an impaired fasting glucose which mimics type 2 diabetes mellitus. In rare cases there is episodic hypotension and rapid cyclic fluctuations of hypertension and hypotension.

Initial Evaluation

The diagnosis of pheochromocytoma is made upon biochemical testing for catecholamine hypersecretion, followed by imaging studies to identify an adrenal tumor. There are many indications for testing that a physician may take into consideration before subjecting patients to many tests and appointments. Some of the indications for testing include the classic triad of symptoms (headache, sweating, and tachycardia), hyperadrenergic spells (palpitations, diaphoresis, tremor, pallor), onset of paroxysmal hypertension or primary hypertension at an early age, or any familial syndromes or history of pheochromocytoma.

Biochemical Testing

The range of biochemical testing that is performed is based upon the index of suspicion that the patient in fact has a pheochromocytoma. Low index of suspicion includes a 24-hour urinary fractionated catecholamines and metanephrines. If there is a high index of suspicion, it is recommended to use a plasma fractionated metanephrines. These tests are performed by high-performance liquid chromatography (HPLC) with tandem mass spectroscopy or electrochemical detection. The more recent techniques have overcome the traditional problems that are associated with drug interference and contrast agents.

Catecholamines

Catecholamines are an organic compound that are derived from the amino acid tyrosine. Tyrosine can either be derived from diet or synthesized from phenylalanine. Epinephrine, norepinephrine, and dopamine are the primary catecholamines that are secreted from the adrenal medulla during the sympathetic flight-or-fight response.

Norepinephrine is a neuromodulator and a hormone that circulates in the blood. Its primary function is to mobilize the brain and the body for action as part of the peripheral sympathetic system. In the flight-or-fight response norepinephrine causes arousal and alertness. It enhances the formation and retrieval of memory, and focuses attention. As a hormone it increases cardiac output by increasing the heart rate and blood pressure. It triggers glycolysis and increases vascular blood flow to the skeletal muscles.

Dopamine functions as a neurotransmitter that is released by neurons to send signals to other functioning nerve synapses. Dopamine plays a critical role in reward-motivated behavior. The anticipation of most types of rewards increase the levels of dopamine in the brain. Many addictive drugs mimic this pathway while simultaneously blocking the reuptake of it. Dopamine is also functional in the motor control pathway as a neuromodulator which controls the release of many other hormones. In circulation outside of the brain, dopamine functions as a chemical messenger. It inhibits norepinephrine release and acts as a vasodilator. It increases renal excretion of sodium. It acts to reduce insulin secretion and gastrointestinal motility.

Epinephrine, also known as adrenaline or adrenalin functions as a neurotransmitter, hormone, and as a medication. It plays an important role in increasing cardiac output, pupil dilation, and increasing insulin release to stimulate glycolysis. It acts on the alpha and beta receptors to ultimate activate the flight-or-fight response. Epinephrine is also used medically to treat a number of conditions including anaphylaxis, cardiac arrest, and bleeding.