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.

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Biotin Interference on Diagnostic Testing

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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.