The Precipitin Reactions


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This figure shows 2 sets of 4 antigens. Each antigen has multiple unique epitopes on the surface. For one set of antigens, polyclonal antibodies are binding to different sets of epitopes on each antigen, linking the antigens together like a lattice. For the second set of antigens, monoclonal antibodies bind to just one of the specific unique epitopes. Because of the specificity, only 2 antigens are linked to each other, rather than forming a more complex lattice with all 4 antigens.
Polyclonal antiserum binds to multiple epitopes on an antigen, leading to lattice formation that results in a visible precipitin. Monoclonal antibodies can only bind to a single epitope; therefore, less binding occurs and lattice formation generally does not occur.

Source: OpenStax Microbiology

OpenStax Microbiology

A visible antigen-antibody complex is called a precipitin, and in vitro assays that produce a precipitin are called precipitin reactions. A precipitin reaction typically involves adding soluble antigens to a test tube containing a solution of antibodies. Each antibody has two arms, each of which can bind to an epitope. When an antibody binds to two antigens, the two antigens become bound together by the antibody. A lattice can form as antibodies bind more and more antigens together, resulting in a precipitin. Most precipitin tests use a polyclonal antiserum rather than monoclonal antibodies because polyclonal antibodies can bind to multiple epitopes, making lattice formation more likely. Although mAbs may bind some antigens, the binding will occur less often, making it much less likely that a visible precipitin will form.

The amount of precipitation also depends on several other factors. For example, precipitation is enhanced when the antibodies have a high affinity for the antigen. While most antibodies bind antigen with high affinity, even high-affinity binding uses relatively weak noncovalent bonds, so that individual interactions will often break and new interactions will occur.

In addition, for precipitin formation to be visible, there must be an optimal ratio of antibody to antigen. The optimal ratio is not likely to be a 1:1 antigen-to-antibody ratio; it can vary dramatically, depending on the number of epitopes on the antigen and the class of antibody. Some antigens may have only one or two epitopes recognized by the antiserum, whereas other antigens may have many different epitopes and/or multiple instances of the same epitope on a single antigen molecule.

The image below illustrates how the ratio of antigen and antibody affects the amount of precipitation. To achieve the optimal ratio, antigen is slowly added to a solution containing antibodies, and the amount of precipitin is determined qualitatively. Initially, there is not enough antigen to produce visible lattice formation; this is called the zone of antibody excess. As more antigen is added, the reaction enters the equivalence zone (or zone of equivalence), where both the optimal antigen-antibody interaction and maximal precipitation occur. If even more antigen were added, the amount of antigen would become excessive and actually cause the amount of precipitation to decline.

(A) Diagram of polyclonal antiserum. Antigens with multiple epitopes (shapes on their surface) are bound to different antibodies (each antibody binds to a different epitope). B) Diagram of monoclonal antibodies. Antigens with multiple epitopes have only one type of antibody bound to a single epitope on each. A graph; the X-axis is labeled antigen added and Y-axis is labeled precipitin formed. In the zone of antibody excess there is more antibody than antigen. In this case, there is no precipitate. In the equivalence zone there are approximately equal amounts of antigen and antibody. In this case a precipitate does form. In the zone of antigen excess, there is more antigen than antibody and no precipitate forms.
As antigen is slowly added to a solution containing a constant amount antibody, the amount of precipitin increases as the antibody-to-antigen ratio approaches the equivalence zone and decreases once the proportion of antigen exceeds the optimal ratio.

Source: OpenStax Microbiology

Source:

Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/microbiology