The Central Dogma of Life

All cells within a complex multicellular organism such as a human being contain DNA. All the DNA together make up that organisms genome. There are many different types of cells within a complex organism. What, then makes a cardiomyocyte different than a hepatocyte? The answer lies within how each cell controls its genome. DNA consists of genes, which are short sequences of nucleic acids that code for particular molecular structures or protein that carries out a specialized function. Each cell can control its unique set of genes. Some are expressed, and others are repressed. This dictates cellular morphology and function. That is how a myocyte differs from a hepatocyte. Its not the DNA itself. All cells contain the same DNA. Rather, it’s how each cell is individualized, and controls how it uses the set of DNA. This expression and repression is highly regulated by cues both within and extrinsic to the cell. This article will serve to cover the first part of what DNA actually is and how it codes for specific polypeptides that carry out downstream functions. This is the central dogma of life.

Nucleic Acids

Nucleic acids are what transfers genetic material as well as participate in cell signaling and other metabolic processes. Often considered the building blocks of cells. There are two main categories of nucleic acids; Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Both DNA and RNA are polymers of nucleotides. Both RNA and DNA structures are similar in that they consist of a sugar (either Ribose, or deoxyribose) with a nitrogen containing base (either a pyrimidine or purine) bonded to a phosphate group, except that DNA does not have a hydroxide group at Carbon-2, where as RNA does.

Bases

Nitrogenous bases are categorized as either a pyrimidine or a purine. A pyrimidine is a heterocyclic aromatic ring structure, whereas a purine is a two ringed heterocyclic aromatic ring. The pyrimidines are Uracil, which only exists in RNA, thymine, which only exists in DNA, and cytosine which coexists. The purines are adenine and guanine, which coexist in both DNA and RNA. Base-pairing is what creates the characteristic double-helix feature of DNA, and the single-stranded RNA structure. Each base-pair consists of one pyrimidine and one purine which are called base complements. In DNA, thymine always binds adenine (T-A), and guanine always binds to cytosine (G-C) through double or triple hydrogen bonds.

DNA molecule

DNA being a double helix is proven to be advantageous in multiple ways; the nitrogenous base which contains the nucleic information is locked within the complex, facing each other in the centre of the molecule, as opposed to in RNA, where the nucleic acid base is exposed to the cellular environment which provides more opportunity for it to be mutated. DNA is more chemically stable than RNA and less susceptible to degradation. Having two complementary strands allows for greater proof-reading mechanisms. Thymine is much more stable than uracil (RNA). Due to deamination cytosine is often changed to uracil, which in DNA is quickly corrected because uracil is not supposed to be there. In RNA, it’s impossible for the cell to know if it’s truly supposed to be there or not.

DNA replication

DNA replication is the biological process of producing two identical copies of DNA from a single original DNA molecule. This process occurs in all living organisms. It beings at specific locations within the genome called origins of replication. These origins of replication are areas of high T-A base pairing. This is important because there are less hydrogen bonds between the A-T bases so the strand is easier to break at those points. This is known as initiation and at this point the DNA strand is unwound by proteins and enzymes known as helicases, which expose the two strands and result in replication forks that are bi-directional from the origin. Topoisomerases are enzymes used to temporarily break the strands of DNA to relieve tension. These two strands then serve as a template for the leading and lagging strands which will be created as a DNA polymerase will match complementary nucleotides to the templates. DNA is always synthesized in the 5′ to 3′ direction and because at the replication fork the template strands are oriented in opposite directions the leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork and replication is continuous in the 5′ to 3′ direction. Within the lagging strand the nascent DNA being synthesized is in the opposite direction, and because of this replication lags and is fragile. Primase is an enzyme that synthesizes a short RNA primer with a free 3′ OH group which is then elongated by a DNA polymerase. The leading strand only receives one RNA primer, while the lagging strand receives several. The leading strand is continuously extended by a DNA polymerase, while the lagging strand is extended discontinuously from the multiple primers forming Okazaki fragments. DNA clamp proteins form a sliding clamp around the DNA, this allows the DNA polymerase to maintain constant contact with the template strands, and enhancing processivity. Once DNA polymerase reaches the end of the template strands and runs into double-strand DNA the sliding clamp protein complex undergoes a conformational change and releases the DNA polymerase. RNase then removes the RNA primers and is replaced by DNA ligase which joins together the multiple parts of the DNA.

DNA replication occurs at multiple points within the chromosome of an organism and because of the linear nature of chromosomes these replication forks are unable to reach the terminal ends and as a consequence a small amount of DNA is lost each replication cycle. Telomeres are regions of repetitive DNA close to the ends of the chromosomes that help mitigate the loss of genomic material during replication. These telomeres are finite and thus each cell has a terminal number of replications. This is otherwise known as the Hayflick limit. When DNA is passed down the germ cell line telomerase is an enzyme that extends the repetitive sequences of the telomere region. Telomerase can become mistakenly up-regulated in somatic cells, which can sometimes lead to cancer.


This is the first release of a 3 part series of articles dedicated to the Central Dogma of life. This article covered what RNA, DNA and DNA replication are.

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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

The Antibody

An antibody or immunoglobulin is a large Y-shaped protein produced primarily by plasma cells of the humoral immune system. They are used to recognize and neutralize any foreign antigens or pathogens. An antibody is identical to the B-cell receptor of the cell that secretes it except for a small portion of the C-terminus of the heavy-chain constant region. The difference is that a B-cell receptor C-terminus is a hydrophobic membrane-anchoring sequence and on an antibody, the C-terminus is a hydrophilic sequence that allows its secretion. The Y-portion of the consists of two arms that vary between the different antibody molecules, otherwise known as the V-region. The V-region is involved in antigen binding. The C-region is far less variable and is the part of the molecule that interacts with effector cells and other molecules. All antibodies are constructed in the same way paired from heavy and light polypeptide chains joined by disulfide bonds so that each heavy chain is linked to a light chain and the two heavy chains are linked together.

There are two types of light chains, lambda and kappa. A given immunoglobulin has one or the either, never both. In humans the ratio of kappa to lambda; the two types of light chains in immunoglobulins is 2:1. The class, and the effector function of an antibody is defined by the structure of its heavy chain. There are five main heavy-chain isotypes. The five major immunoglobulin classes are IgM, IgD, IgG, IgA, and IgE. IgG is the most abundant immunoglobulin and has several subclasses (1, 2, 3, and 4 in humans). The distinctive functional properties are conferred by the carboxyl -terminal part of the heavy chain, where it is not bonded with the heavy chain.

Each chain of the immunoglobulin consists of a protein domain. Each protein domain consists of a series of similar, but not identical sequences about 110 amino acids long . The light chain is made up of two domains, and the heavy chain consists of four. The variable or V-domain of the heavy and light chains together consist of the V-region of the antibody allowing it to bind specific antigens. The constant domains of the heavy and light chains together make up the C-region. The V-region or the Y of the molecule, where the antigen binding activity takes place is called the Fab fragments. Fab stands for fragment antigen binding. The other part of the molecule, the constant region (C-region) contains no antigen-binding activity, and is called the Fc fragment. Fc stands for Fragment crystallizable. This is the part of the molecule that interacts with effector molecules and cells.

The immunoglobulin molecule is flexible. There is a hinge region that links the Fc and Fab regions of the molecule, allowing independent movement of the two Fab arms.

Recap

To recap. An antibody molecule is made up of four polypeptide chains, comprising of two identical light chains and two identical heavy chains, which can be thought of as forming a flexible Y-shaped structure. Each of the four chains has a variable (V) region at its amino terminus, which contributes to the antigen-binding site, and a constant (C) region, which determines the isotype of the immunoglobulin. The light chains are bound to the heavy chains are non-convalent disulfide bonds. The V-regions of the light and heavy chains pair together to form the Fab region on the arms of the Y-structure. The trunk of the Y-structure, consisting of the carboxyl-terminal domains of the heavy chains make up the Fc fragment. The Fc fragment determines the different isotype of the immunoglobulin and interacts with different effector molecules. There is a hinge region joining the Fab and Fc regions allowing the antibody independent movement to maximize its antigen binding capabilities.

 

 

 

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.

Cells Cells Cells!

The immune system is the host defense system against foreign pathogens. It is an extremely adept system comprised of the innate immune system and the adaptive immune system, as well as complement. For more information on those two systems as a whole, review part one of the immune system.

This part of the immune system overview will focus on the leukocytes or granulocytes of the innate immune system.

The most abundant leukocyte is the neutrophil. It comprises about 40-70% of the white cells an individual has. The maturation of the neutrophil is myeloblast, promyelocyte, myelocyte, metamyelocyte, band neutrophil, segmented neutrophil. The cytokine responsible for stimulating neutrophil production in the bone marrow is G-CSF (granulocyte colony stimulating factor). There are three pools in the bone marrow, the stem cell pool, consisting of HSCs, the proliferative pool, full of mitotic cells, and the maturation (storage) pool. Full of metamyelocytes, bands, and PMNs. During the proliferative pool stage GM-CSF, G-CSF and IL-3 are all used as growth factors to help the neutrophil differentiate and mature. Granulocyte release from the bone marrow is stimulated by G-CSF. Once in circulation neutrophils are divided randomly into either a circulating pool and a marginated pool. The neutrophils in the marginated pool are loosely localized to the walls of capillaries in tissues. Neutrophils can move freely between the two pools. Integrins and selectins are important as they allow neutrophils to marginate and allow them to move into the tissues by using diapedesis. Diapedesis is the extravasation of blood cells through intact vessel walls.

In response to inflammatory mediators and chemoattractants the neutrophil is activated and it results in reorganization of the actin cytoskeleton, membrane ruffling, adhesion and motility. In basic terms, when an infection is ongoing, the surrounding cells and tissue release cytokines and inflammatory mediators that attract neutrophils specifically to come to the site and help control the infection. Chemotaxis is the term for this. The neutrophil attaches to the substratum (endothelial surface) which allows extensions of pseudopods to attach through integrins. Contraction allows the cell body to be pulled forward (still attached). Release of the neutrophil at the back allows the cell to move forward.

Once the neutrophil has reached the site of infection it aids in fighting the infection or pathogen by phagocytosis. Phagocytosis occurs when a neutrophil surface receptor recognizes an antigen either through direct recognition, or to recognize an opsonized antigen. An opsonized antigen remember is when particular cellular processes, such as complement, present pathogenic antigens to these neutrophils to aid in phagocytosis so the neutrophil doesn’t have to search for the pathogen. With recognition comes attachment and engulfment. Cytoplasmic pseudopodia surround the particle forming a phagosome within the neutrophil cytoplasm. The formation of the phagosome allows the NADPH oxidase complex to form which leads to the generation of reactive oxygen species (ROS) such as hydrogen peroxide which is converted to hypochlorite by myeloperoxidase. (O2 dependent). A series of metabolic changes can occur like the changing of the pH and that allows primary or secondary granules within the neutrophil to release numerous bactericidal molecules into the phagosome. (O2-Independent). Bactericidal molecules aid in the killing of foreign pathogens. There is a third mechanism to which neutrophils are able to fight off foreign invader and its by using NETS. Neutrophils can generate an extracellular net that consists of chains of nucleosomes from unfolded nuclear chromatin. These structures have enzymes from neutrophil granules and can trap and kill some gram positive and gram negative bacteria, and fungi. NETs are generated at the time neutrophils die.

Monocyte development is similar to that of neutrophilic maturation because they are both derived from the granulocyte monocyte progenitor. M-CSF (macrophage colony stimulating factor) is the major cytokine responsible for the growth and differentiation of monocytes. Once in the tissue, monocytes differentiate into macrophages, depending on the tissue that the monocytes migrate too, for example, in the lymph nodes they differentiate into dendritic cells, and in the liver, they differentiate into Kupffer cells.

Eosinophils make up 1-3% of the cells in the bone marrow. Eosinophil granules are full of synthesized proteins, cytokines, chemokines, growth factors and cationic proteins. Degranulation can occur in multiple ways; by classic exocytosis, granules move to and fuse with the plasma membrane and the granules secrete into the ECS (Extracellular Space). By compound exocytosis, the granules fuse in the cytoplasm before moving to the plasma membrane. Piecemeal degranulation is when vesicles remove specific proteins from the secondary granules and then migrate to the plasma membrane and then emptying into the ECS. Eosinophils play a role in immune regulation. Eosinophils secrete major basic protein (MBP) which is the cause of mast cell degranulation and cytokine production. Eosinophils are implicated in both type 1 and type 2 immune response, primarily being infectious diseases. Eosinophils are primarily implicated in parasitic infections and are the hallmark characteristic of helminth infections. They help drive antibody production and suppress phagocytosis by secreting arylsufatase which inactivated leukotrienes and secrete antihistamine which counteracts the action of mast cells and basophils.

Basophils and Mast cells are usually grouped together, although basophils are a true WBC because they mature in the bone marrow and circulate in the blood with granules. Mast cell precursors leave the bone marrow and migrate to a tissue where they mature. Basophils and mast cells have membrane bound IgE on their surface. When activated by an antigen causes degranulation (histamine and heparin, which leads to an inflammation causing vasodilation and edema. They also secrete cytokines that activate B and T cells. Basophils are capable of releasing large quantities of subtype 2 helper T cell cytokines such as IL-4, and IL-13 that regulate the TH2 immune response. Mast cells function in chronic allergic reactions, Basophils are the initiators of allergic inflammation through the release of preformed cytokines. Basophils can play a rule in angiogenesis through the release of VEGF and its receptors.

To recap everything that has been learned with this article; neutrophils are the most abundant leukocyte encountered and play a huge role in the innate immune system. They are often the first to a site of infection or inflammation and use multiple mechanics of phagocytosis to control the situation. Monocytes circulate and settle into a tissue where they become resident macrophages. Macrophages also phagocytize foreign antigens when it comes into contact with the tissue they reside in. Eosinophils are active in infections, particularly of parasitic origin. Eosinophilia is a common finding in helminth infections. Basophils and mast cells work in conjunction with IgE to mediate hypersensitivity allergic reactions. They are the ones to thank for making your nose run.