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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Overview of the Immune System; Part One

The overall function of the immune system is to prevent or limit infection. It is essential for survival. Multiple organ systems, cells, and proteins are involved in the immune response. It is the most complex system that the human body has. The immune system is differentiated into two directions. Innate or non-specific immunity or Acquired (specific) immunity.

The Innate immune system consists of many components. The skin acts as a mechanical barrier and is typically the first line of defense against foreign substances. Mucous membranes consist of the bodies normal microbiota which compete with invading microbes. The mucous membranes are also lined with mucous and cilia which act in an elevator type motion to push foreign substances away. Physiological barriers such as temperature, pH and the complement system. The more acidic environment that a lower pH offers disrupts bacterial growth. Antimicrobial proteins and peptides are present in different epithelial locations in the body. Lysozymes are present in the tears and saliva and cleave the peptidoglycan cell wall present in bacteria. Secretory phospholipase A2 is present in the gut and can enter the bacterial cell and hydrolyze lipids in the cell membrane. Lectins target gram positive bacteria and forms pores in the membranes. Defensins integrate into the lipid and form pores which causes loss of membrane integrity. These defensins are present in PMNs (neutrophils) and lamellar bodies in the gut. Cathelicidins are present in neutrophils and macrophages in the lungs and intestines and distrupt membranes. Histatins are constitutively produced by the glands in the oral cavity and are active against pathogenic fungi.  Inflammation plays a huge role in the Innate immune system. Inflammation induces vasodilation and increase in capillary permeability causing an influx of immune cells like PMNs and macrophages. Inflammation can be observed by the four cardinal signs; rumor (redness), tumor (swelling), color (heat), and dolor (pain). The innate immune response is a rapid response.

Innate Immunity

The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b. There are three distinct pathways; the classical pathway, the lectin pathway, and the alternative pathway. All pathways generate a C3 convertase which cleaves C3, leaving C3b bound to the microbial surface and releasing C3a. In the classical pathway the activated C1s cleaves C4 to C4a and C4b which binds to the microbial surface. C4b then binds C2, which is cleaved by C1s to C2a and C2b forming the C4b2b complex. C4b2b on the microbial surface is an active C3 convertase which cleaves C3 to C3a and C3b. This results in opsonization of the bacterial surface by C3b. The C4b2b3b complex is an active C5 convertase leading to the development of the membrane-attack complex. Each complement component (C4a/b, C2a/b, C3a/b) have different functions, but that is another discussion for another time. The lectin pathway of complement activation is when mannose-binding lectin (MBL) and ficolins recognize and bind to carbohydrates on the pathogen surface. Ficolins are similar to MBLs, but have a different carbohydrate binding domain. MBLs bind with high affinity to mannose and fucose residues. Conversely ficolins bind oligosaccharides containing acetylated sugars. When MBL binds to a pathogen surface MBL-associated serine protease (MASP)-2 is activated and cleaves C4 and C2 similar to the classical pathway. The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin (factor P) in the presence of pathogens. Properdin stabilizes the C3bBb complex. C3 undergoes spontaneous hydrolysis to C3(H20) which binds to factor B, allowing it to be cleaved by factor D into Ba and Bb. The C3(H20)Bb complex is essentially a C3 convertase which cleaves more C3 into C3a and C3b. C3b molecules result in opsonization of bacterial surfaces. Its important to recognize that all pathways lead to generation of a C5 convertase. C4b2a4b in the classical pathway, C4b2a3b in the lectin pathway, and C3b2Bb in the alternative pathway. C5 is cleaved into C5a/b that initiates the assembly of the terminal complement components. These are the terminal complement components that form the membrane-attack complex.

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The membrane attack complex consists of an assembly of C6, C7, and C8. This complex undergoes a conformational change that results in polymerization of C9 which generates a large pore in the cell membrane. Host cells contain CD59 which prevents the assembly of the C9 molecules preventing the formation of the membrane-attack complex.

C3a, C4a, and C5a are unique in that these complement components are called anaphylatoxics. They initiate a local inflammatory response when systemic injection of these molecules occurs. They induce smooth muscle cell contraction and increased vascular permeability. They induce adhesion molecules and activate mast cells that invade and populate submucosal tissues to release inflammatory mediators such as histamine and TNF-a.

The Acquired or adaptive immune system is all about specificity. The Humoral branch of the acquired immune system is executed by the B lymphocytes that produce antibodies to specific antigens. The cell-mediated branch consists of antigen presenting cells (APC) such as the dendritic cells processing foreign substances and presenting proteins of those substances as antigens through the major histocompatibility complex (MHC) to CD8 T lymphocytes. These are cytotoxic T-cells that kill these foreign antigens. The acquired immune response is a slow response because it takes the body time to produce antibodies. An important aspect of the adaptive response is memory. Once antibodies have been produced to an antigen, these responses last and the time it takes to produce an antibody on subsequent exposures is rapidly decreased.

These two different systems work in conjunction to produce an adequate and sustained response. When foreign antigens are processed and expressed on the surface of APCs as MHC peptides, pro-inflammatory cytokines such as IL-12p70, IL-18, and IFN-a are secreted. These attract NK cells which primarily attack viruses as well as PMNs and macrophages that phagocytize these antigen peptides to destroy them. Adaptive immunity is also started with dendritic cells that also undergo antigen uptake and processing. This is also called the maturation signal. This signal is augmented by IFN-y and TNF-a secreted by macrophages and NK cells. These dendritic cells either present the antigen to B lymphocytes which are the antibody producers or they present the antigen to CD4/CD8 T-cell lymphocytes.

There are multiple classes of antibodies. IgD is typically expressed on B-cell lymphocytes during differentiation with IgM. IgD is also present in the serum in low concentrations. IgM is a pentamer and the largest immunoglobulin. It is the first antibody that is produced in the immune response. IgA is in high concentration in the mucosal linings, saliva, and tears. Typically part of first line defenses. IgG is present in high concentrations in the serum. IgG is unique in that it can cross the placenta. IgE is involved in allergic reactions. It binds to mast cells and basophils causing degranulation.

-Caleb