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Hapten are small-molecules, that can only become immunogenic when conjugated with a carrier protein. I was wondering if all small-molecules can become haptens (eg. by synthetic conjugation). Given that lymphocytes are filtered by negative selection, I would expect that small-molecules that chemically mimic self-peptides would not work as haptens (at least not in the sense, that they could be detected by antibodies).
Are there any examples of small-molecules that have been tried as haptens but could not generate antibodies ?
Also is there any estimate of the ratio of naturally occuring haptens and synthetic haptens. I was investigating the IEDB (https://www.iedb.org/) as well as SuperHapten (http://bioinformatics.charite.de/superhapten/) but could not find a way to filter these two types of haptens.
14.2.1: Architecture of the Immune System
- Contributed by OpenStax
- General Biology at OpenStax CNX
- Define memory, primary response, secondary response, and specificity
- Distinguish between humoral and cellular immunity
- Differentiate between antigens, epitopes, and haptens
- Describe the structure and function of antibodies and distinguish between the different classes of antibodies
Olivia, a one-year old infant, is brought to the emergency room by her parents, who report her symptoms: excessive crying, irritability, sensitivity to light, unusual lethargy, and vomiting. A physician feels swollen lymph nodes in Olivia&rsquos throat and armpits. In addition, the area of the abdomen over the spleen is swollen and tender.
- What do these symptoms suggest?
- What tests might be ordered to try to diagnose the problem?
Adaptive immunity is defined by two important characteristics: specificity and memory. Specificity refers to the adaptive immune system&rsquos ability to target specific pathogens, and memory refers to its ability to quickly respond to pathogens to which it has previously been exposed. For example, when an individual recovers from chickenpox, the body develops a memory of the infection that will specifically protect it from the causative agent, the varicella-zoster virus, if it is exposed to the virus again later.
Specificity and memory are achieved by essentially programming certain cells involved in the immune response to respond rapidly to subsequent exposures of the pathogen. This programming occurs as a result of the first exposure to a pathogen or vaccine, which triggers a primary response. Subsequent exposures result in a secondary response that is faster and stronger as a result of the body&rsquos memory of the first exposure (Figure (PageIndex<1>)). This secondary response, however, is specific to the pathogen in question. For example, exposure to one virus (e.g., varicella-zoster virus) will not provide protection against other viral diseases (e.g., measles, mumps, or polio).
Adaptive specific immunity involves the actions of two distinct cell types: B lymphocytes (B cells) and T lymphocytes(T cells). Although B cells and T cells arise from a common hematopoietic stem cell differentiation pathway, their sites of maturation and their roles in adaptive immunity are very different.
B cells mature in the bone marrow and are responsible for the production of glycoproteins called antibodies, or immunoglobulins. Antibodies are involved in the body&rsquos defense against pathogens and toxins in the extracellular environment. Mechanisms of adaptive specific immunity that involve B cells and antibody production are referred to as humoral immunity. The maturation of T cells occurs in the thymus. T cells function as the central orchestrator of both innate and adaptive immune responses. They are also responsible for destruction of cells infected with intracellular pathogens. The targeting and destruction of intracellular pathogens by T cells is called cell-mediated immunity, or cellular immunity.
Figure (PageIndex<1>): This graph illustrates the primary and secondary immune responses related to antibody production after an initial and secondary exposure to an antigen. Notice that the secondary response is faster and provides a much higher concentration of antibody.
- List the two defining characteristics of adaptive immunity.
- Explain the difference between a primary and secondary immune response.
- How do humoral and cellular immunity differ?
Hypersensitivity Types: 4 Important Types of Hypersensitivity
The following points highlight the four important types of hypersensitivity. The types are: 1. Type I Hypersensitivity (Anaphylaxis) 2. Type II Hypersensitivity (Cytotoxic Hypersensitivity) 3. Type III Hypersensitivity 4. Type IV Hypersensitivity.
1. Type I Hypersensitivity (Anaphylaxis):
This type of hypersensitivity is the most common among all the types. About 17% of the human population may be affected, probably due to a natural proneness controlled by the genetic make-up. Anaphylaxis which literally means “opposite of protection” — is mediated by IgE antibodies through interaction with an allergen.
The allergens inciting anaphylaxis include a great variety of substances, like pollens, fibres, insect, venom, fungal spores, house-dust etc. as well as various food materials like egg, milk, fish, crab-meat, peanuts, soybean, various vegetables etc.
Generally, anaphylactic responses are of a mild type producing symptoms, like hay-fever, running nose, skin-eruptions known as “hives” or breathing difficulties. But in some cases, the responses may be severe and may even prove fatal. This latter type of response is called anaphylactic shock.
This may develop within a few minutes (2 to 30 min) and may cause death before any medical help can be provided. Anaphylactic shock is known to result from a bee-sting or intramuscular injection of penicillin. Penicillin itself is not an antigen, but it can act as a hapten.
After combination with serum proteins, it can stimulate allergenic response producing IgE molecules which can combine with the drug. A person sensitized with penicillin may be a victim to anaphylactic shock. The severe form of anaphylaxis is considered as systemic, in contrast to the milder forms which are localized.
During the sensitization phase, the immune system produces B-lymphocytes which are transformed into plasma cells in the usual way. But the plasma cells produce IgE antibodies complementary to the allergenic antigen, instead of normal IgG and IgM antibodies.
The IgE antibodies, so produced, circulate in the blood stream and they become attached to the mast cells and basophils, because IgE antibodies have special affinity for these cells. Mast cells and basophils have a richly granular cytoplasm and each cell has numerous (>100,000) binding sites for IgE molecules.
The IgE antibodies bind to these cells with their Fc domain, while the antigen-binding sites remain free. The sensitization period takes about a week’s time to be completed. During this period millions of IgE molecules are produced and fixed on the mast cells and basophils.
Manifestation of anaphylactic symptoms appears when such a sensitized person is exposed to the same allergen again. The allergen entering into body reacts with the its complementary IgE molecules bound to mast cells and basophils and combine with the antigen binding sites of the antibody.
This interaction causes degranulation of mast cells and basophils and the granules are released in the body fluids. Mast cells occur in close association with the capillaries throughout the body, particularly in the skin and respiratory tracts.
The granules released by mast cells and basophils contain several preformed chemical mediators of which the most important is histamine. The others include heparin, serotonin, bradykinin etc. In addition, some secondary mediators are also produced as a result of the interaction between IgE and an allergen. They include the leucotrienes and prostaglandins.
Degranulation of mast cells and basophils occurs when two IgE molecules are adjacent to each other on these cells and both bind to an antigen (allergen) having the same specificity, thereby forming a bridge. The chemical mediators released by the granules produce various changes associated with allergic response. One of the most important effect is the contraction of smooth muscles.
The small veins are constricted and capillary pores are dilated leading to extrascular accumulation of fluid (edema). The bronchial muscles, as well as those of GI tract, may also contract producing breathing difficulty and cramps. Mast cells present in the mucous membrane of the upper and lower respiratory tracts cause rhinitis and asthma. The events occurring during the sensitization phase and the expression of allergic symptoms after a second encounter with the allergen are diagrammatically shown in Fig. 10.58.
The susceptibility to specific allergens of an individual can be determined by skin-test. It is performed by injecting a small amount of the possible allergens below the skin. A wheal and erythema response indicated by itching, swelling and reddening of the injection spot developed within 2 to 3 minutes and reaching a maximum in about 10 minutes means that the substance is allergenic. Avoidance of the identified allergen(s) is the best way of prevention of anaphylaxis.
Another way of prevention is by desensitization. Once an allergen has been identified, the sensitive person is injected with small doses of the allergen for several weeks. The objective is to build­up immunity to the allergen through production of excess of IgG antibodies, so that they outnumber IgE antibodies. The IgG antibodies in this case are called blocking antibodies, because they block the IgE antibodies to combine with the allergen.
Anaphylaxis can also be prevented by specific drugs. Anaphylactic shock can be prevented by immediate injection of epinephrine. Drugs used for localized anaphylaxis act in two ways. A group of drugs like dexamethasone, prednisolone etc. inhibit the production or release of the chemical mediators responsible for development of allergic symptoms. The other group, mainly the anti-histamines, inhibit the action of chemical mediators, mainly that of histamine.
2. Type II Hypersensitivity (Cytotoxic Hypersensitivity):
This type of hypersensitivity involves IgG antibodies and the complement system and results in cell destruction. IgM may also take part in cell damaging reactions. Cytotoxic hypersensitivity is the result of transfusion of incompatible blood of a donor to a recipient, although this is of rare occurrence because of careful cross-matching of the donor and the recipient’s blood-groups.
A faulty cross­-matching leads to hemolysis of the donor’s erythrocytes in the blood vessels of the recipient. This happens because the alloantigen’s of the donor’s erythrocytes react with the antibodies in the serum of the recipient and in combination with activated complement, the erythrocytes undergo hemolysis.
Similarly, when an Rh-negative recipient is transfused with the blood of an Rh-positive donor, Rh-antibodies develop in the recipient. In case, the same recipient receives subsequently blood from an Rh-positive donor, a rapid and extensive hemolysis occurs in the recipient due to interaction of the Rh-antigen and Rh-antibody. Precaution is necessary that an Rh-negative recipient is not transfused with Rh-positive blood more than once.
Interaction of Rh-antigen and Rh-antibody may lead to a more serious consequence when an Rh-negative mother bears an Rh-positive child, the trait of the child being acquired from an Rh-positive father. Rh-antigen of the fetus enters into mother’s circulation and provokes formation of Rh-antibody in mother.
In a succeeding pregnancy resulting in an Rh-positive fetus, these antibodies enter into the fetal circulation through placenta and react with the Rh-antigen producing serious complications, known as haemolytic disease of the newborn.
The situation leading to this disease is diagrammatically shown in Fig. 10.59:
3. Type III Hypersensitivity:
Normally, the antigen-antibody complex formed as a result of immune reactions is removed by the phagocytic activity of body. However, when bulky antigen-antibody complexes are formed and the aggregates combine with the activated complement, they chemotactically attract the polymorphonuclear leucocytes. These cells release lysosomal enzymes in large quantities to Cause tissue damage. This results in immune complex hypersensitivity (Type III hypersensitivity).
One form of this type of hypersensitivity is the Arthus Reaction. It develops due to deposition of IgG-antigen complexes in the blood vessels causing local damage. When such aggregates are deposited in blood vessels of kidney glomeruli, the result may be nephritis.
Similarly, inhalation of bacteria and fungal spores may give rise to a disease called farmer’s lung. The antigens react with IgG antibodies to form complexes in the epithelial layers of the respiratory tract giving rise to this ailment.
Another form of this type of hypersensitivity is known as lupus (systemic lupus erythematosus). It is produced as a result of interaction of IgG and the nucleoproteins of the disintegrated leucocytes (auto-antigens). Therefore, lupus is an autoimmune disease. The immune-complex may be deposited locally in the skin, or systemically in kidney or heart. Rheumatoid arthritis is another autoimmune disease developing from deposition of immune complexes in the joints.
Serum sickness is another manifestation of immune complex hypersensitivity. Antisera like anti-tetanus serum (ATS) may act as antigen in human body, because these are obtained from animals and are injected to persons for providing immediate protection. The antigen (ATS, for example) can provoke an immune response to produce IgG in the body. These IgG antibodies react with the antisera to produce immune complexes and give rise to serum sickness.
Immune complex hypersensitivity (Type III) is diagrammatically shown in Fig. 10.60:
4. Type IV Hypersensitivity:
In contrast to the first three types of hypersensitivity, Type IV is mediated by cells of immune system, mainly T-cells, but also macrophages and dendritic cells. Furthermore, lymphokines produced by T-cells play an important role. The expression of allergic manifestations takes a longer time, at least 24 hr or more.
Hence, Type IV hypersensitivity is called delayed type of hypersensitivity. The delay in appearance of allergic symptoms after a second exposure to an allergen is mainly due to the time taken by the cellular components to migrate to the site where antigen is present.
The cells involved in delayed hypersensitivity are mainly T-lymphocytes. T-lymphocytes have two main types, — the CD4+ cells and CD8+ cells. The cells involved in Type IV hypersensitivity belong to the CD4+ type. The special group of CD4+ cells taking part in this hypersensitivity are called TD-cells (D standing for delayed hypersensitivity). TD-cells are a part of the T-helper cell (TH-cells) population which constitutes the bulk of CD4+ T-cells. TH-cells are distinguished into TH-1 and TH-2 types, of which TH-2 cells are mainly responsible for activation of B-cells to produce immunoglobulin’s and TH-1 cells are involved in causing the inflammatory responses including delayed hypersensitivity reactions. So, TD-cells belong to the TH-1 type of lymphocytes.
Like the Type I hypersensitivity, Type IV also has two phases: a sensitization phase and an active phase. The allergen can be a microbial antigen or a small molecule that can act as a hapten and can combine with a tissue protein to form an active antigen. The sensitizing antigen binds to some tissue cells and these are ingested by phagocytic cells, like macrophages and dendritic cells. These cells process the antigen and present the antigenic determinants to the TD-cells.
These T-cells recognize the determinants by interacting with the determinants complexed with MHC proteins of the antigen- presenting cells (APC). The close binding between the T-cells and APCs activates the T-cells to proliferate forming a clone including some memory T-cells. Thereby, the person becomes sensitized to the particular allergenic antigen.
In the next phase, the sensitized individual expresses delayed type of hypersensitivity when exposed at din to the same allergen. The memory T-cells activate the sensitized T-cells to produce lymphokines which cause the inflammatory responses associated with Type IV hypersensitivity.
The whole process is diagrammatically shown in Fig. 10.61:
A well-known example of a microbial agent that elicits a delayed hypersensitivity is tuberculin which is a purified protein derivative (PPD) of tubercle bacilli (Mycobacterium tuberculosis). Other microbial agents that stimulate delayed hypersensitivity are Mycobacterium leprae, Brucella and fungi causing histoplasmosis (Histoplasma capsulatum) and candidiasis (Candida albicans).
The tuberculin skin test (Mantoux test) is used to determine if a person has T-cell mediated reactivity towards tubercle bacilli (also known as Koch’s bacilli). In a sensitized individual, an intradermal injection of 0.1 μg of tuberculin results in development of a progressively increasing swollen reddened circular area at the injection site attaining a maximum size in 24 to 72 hr.
Histologically, the response is due to accumulation of large number of inflammatory cells, mainly lymphocytes and macrophages. A positive response shows that the person has immunity to tuberculosis, developed either through active infection or through vaccination and, therefore, does not require BCG vaccination.
Certain low-molecular weight chemical substances can also evoke delayed hypersensitivity. Generally, the allergic symptoms are restricted to the skin and the response is called contact sensitivity. The clinical manifestation is contact dermatitis. A great many varieties of such agents causing contact dermatitis are known. Some examples are metallic nickel and copper, turpentine, formaldehyde, insecticides, detergents, cosmetics, latex, furs, protein fibres etc.
Certain plants, like poison ivy, poison oak etc. can also provoke contact sensitivity. Detection of the possible sensitizing agent can be made by a patch test in which the suspected agent is kept in contact with skin for 24 hr to 48 hr and the skin reaction is examined. Generally, avoidance of the allergenic substance or material removes the adverse effects promptly.
Hapten
- A substance that is non-immunogenic but which can react with the products of a specific immune response.
- Haptens are small molecules which could never induce an immune response when administered by themselves but which can when coupled to a carrier molecule.
- Haptens have the property of antigenicity but not immunogenicity.
Contents
Nucleic acid Edit
Nucleic acid aptamers are nucleic acid species (next-gen antibody mimics) having selectivity at par of antibodies for a given target generated via in-vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) ranging from small entities such as heavy metal ions to large entities like cells. [1] On the molecular level, aptamers bind to its cognate target through various non-covalent interactions viz., electrostatic interactions, hydrophobic interactions, and induced fitting. Aptamers are useful in biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecule, antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. [2]
In 1990, two labs independently developed the technique of selection: the Gold lab, using the term SELEX for their process of selecting RNA ligands against T4 DNA polymerase and the Szostak lab, coining the term in vitro selection, selecting RNA ligands against various organic dyes. The Szostak lab also coined the term aptamer (from the Latin, apto, meaning 'to fit') for these nucleic acid-based ligands. Two years later, the Szostak lab and Gilead Sciences, independent of one another, used in vitro selection schemes to evolve single stranded DNA ligands for organic dyes and human coagulant, thrombin (see anti-thrombin aptamers), respectively. There does not appear to be any systematic differences between RNA and DNA aptamers, save the greater intrinsic chemical stability of DNA.
The notion of selection in vitro was preceded twenty-plus years prior when Sol Spiegelman used a Qbeta replication system as a way to evolve a self-replicating molecule. [3] In addition, a year before the publishing of in vitro selection and SELEX, Gerald Joyce used a system that he termed 'directed evolution' to alter the cleavage activity of a ribozyme.
Since the discovery of aptamers, many researchers have used aptamer selection as a means for application and discovery. In 2001, the process of in vitro selection was automated [4] [5] [6] by J. Colin Cox in the Ellington lab at the University of Texas at Austin, reducing the duration of a selection experiment from six weeks to three days.
While the process of artificial engineering of nucleic acid ligands is highly interesting to biology and biotechnology, the notion of aptamers in the natural world had yet to be uncovered until 2002 when two groups led by Ronald Breaker and Evgeny Nudler discovered a nucleic acid-based genetic regulatory element (which was named riboswitch) that possesses similar molecular recognition properties to the artificially made aptamers. In addition to the discovery of a new mode of genetic regulation, this adds further credence to the notion of an 'RNA World', a postulated stage in time in the origins of life on Earth.
Both DNA and RNA aptamers show robust binding affinities for various targets. [7] [8] [9] DNA and RNA aptamers have been selected for the same target. These targets include lysozyme, [10] thrombin, [11] human immunodeficiency virus trans-acting responsive element (HIV TAR), [12] hemin, [13] interferon γ, [14] vascular endothelial growth factor (VEGF), [15] prostate specific antigen (PSA), [16] [17] dopamine, [18] and the non-classical oncogene, heat shock factor 1 (HSF1). [19] In the case of lysozyme, HIV TAR, VEGF and dopamine the DNA aptamer is the analog of the RNA aptamer, with thymine replacing uracil. The hemin, thrombin, and interferon γ, DNA and RNA aptamers were selected through independent selections and have unique sequences. Considering that not all DNA analogs of RNA aptamers show functionality, the correlation between DNA and RNA sequence and their structure and function requires further investigation.
Lately, a concept of smart aptamers, and smart ligands in general, has been introduced. It describes aptamers that are selected with pre-defined equilibrium ( K d Recent developments in aptamer-based therapeutics have been rewarded in the form of the first aptamer-based drug approved by the U.S. Food and Drug Administration (FDA) in treatment for age-related macular degeneration (AMD), called Macugen offered by OSI Pharmaceuticals. In addition, the company NeoVentures Biotechnology Inc. [20] has successfully commercialized the first aptamer based diagnostic platform for analysis of mycotoxins in grain. Many contract companies develop aptamers and aptabodies to replace antibodies in research, diagnostic platforms, drug discovery, and therapeutics. Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamer's inherently low molecular weight. Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. An example is a tenascin-binding aptamer under development by Schering AG for cancer imaging. Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, etc. (both of which are used in Macugen, an FDA-approved aptamer) are available to scientists with which to increase the serum half-life of aptamers easily to the day or even week time scale. Another approach to increase the nuclease resistance of aptamers is to develop Spiegelmers, which are composed entirely of an unnatural L-ribonucleic acid backbone. A Spiegelmer of the same sequence has the same binding properties of the corresponding RNA aptamer, except it binds to the mirror image of its target molecule. In addition to the development of aptamer-based therapeutics, many researchers such as the Ellington lab have been developing diagnostic techniques for aptamer based plasma protein profiling called aptamer plasma proteomics. This technology will enable future multi-biomarker protein measurements that can aid diagnostic distinction of disease versus healthy states. Furthermore, the Hirao lab applied a genetic alphabet expansion using an unnatural base pair [21] [22] to SELEX and achieved the generation of high affinity DNA aptamers. [23] Only few hydrophobic unnatural base as a fifth base significantly augment the aptamer affinity to target proteins. As a resource for all in vitro selection and SELEX experiments, the Ellington lab has developed the Aptamer Database cataloging all published experiments. Split aptamers are composed of two or more DNA strands that mimic segments of a larger parent aptamer. The ability of each component strand to bind targets will depend on the location of the nick and the secondary structures of the daughter strands, with the most prominent structures being three-way junctions. [24] The presence of a target molecule can promote assembly of the DNA fragments. [25] Once assembled, the strands can be chemically or enzymatically ligated into a single strand. Analytes for which split aptamers have been developed include the protein α-thrombin, ATP, and cocaine. Split aptamers are a potential template for biosensors in analogy to split protein systems. Peptide aptamers [26] are artificial proteins selected or engineered to bind specific target molecules. These proteins consist of one or more peptide loops of variable sequence displayed by a protein scaffold. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind cellular protein targets and exert biological effects, including interference with the normal protein interactions of their targeted molecules with other proteins. Libraries of peptide aptamers have been used as "mutagens", in studies in which an investigator introduces a library that expresses different peptide aptamers into a cell population, selects for a desired phenotype, and identifies those aptamers that cause the phenotype. The investigator then uses those aptamers as baits, for example in yeast two-hybrid screens to identify the cellular proteins targeted by those aptamers. Such experiments identify particular proteins bound by the aptamers, and protein interactions that the aptamers disrupt, to cause the phenotype. [27] [28] In addition, peptide aptamers derivatized with appropriate functional moieties can cause specific postranslational modification of their target proteins, or change the subcellular localization of the targets. [29] Peptide aptamers can also recognize targets in vitro. They have found use in lieu of antibodies in biosensors [30] [31] and used to detect active isoforms of proteins from populations containing both inactive and active protein forms. [32] Derivatives known as tadpoles, in which peptide aptamer "heads" are covalently linked to unique sequence double-stranded DNA "tails", allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative real-time polymerase chain reaction) of their DNA tails. [33] The peptides that form the aptamer variable regions are synthesized as part of the same polypeptide chain as the scaffold and are constrained at their N and C termini by linkage to it. This double structural constraint decreases the diversity of the conformations that the variable regions can adopt, [34] and this reduction in conformational diversity lowers the entropic cost of molecular binding when interaction with the target causes the variable regions to adopt a single conformation. As a consequence, peptide aptamers can bind their targets tightly, with binding affinities comparable to those shown by antibodies (nanomolar range). Peptide aptamer scaffolds are typically small, ordered, soluble proteins. The first scaffold, [26] which is still widely used, [35] is Escherichia coli thioredoxin, the trxA gene product (TrxA). In these molecules, a single peptide of variable sequence is displayed instead of the Gly-Pro motif in the TrxA -Cys-Gly-Pro-Cys- active site loop. Improvements to TrxA include substitution of serines for the flanking cysteines, which prevents possible formation of a disulfide bond at the base of the loop, introduction of a D26A substitution to reduce oligomerization, and optimization of codons for expression in human cells,. [35] [36] Reviews in 2015 have reported studies using 12 [35] and 20 [37] other scaffolds. Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers. All the peptides panned from combinatorial peptide libraries have been stored in a special database with the name MimoDB. [38] [39] Selection of Ligand Regulated Peptide Aptamers (LiRPAs) has been demonstrated. By displaying 7 amino acid peptides from a novel scaffold protein based on the trimeric FKBP-rapamycin-FRB structure, interaction between the randomized peptide and target molecule can be controlled by the small molecule Rapamycin or non-immunosuppressive analogs. The Affimer protein, an example of a peptide aptamers, is a small, stable protein engineered to display peptide loops that provides a binding surface for a specific target protein. It is a protein of low molecular weight, 12–14 kDa, [40] derived from the cysteine protease inhibitor family of cystatins. [41] [42] [43] [44] The Affimer scaffold is a stable protein based on the cystatin protein fold. It displays two peptide loops that can be randomized to bind different proteins with high affinity. Stabilization of the peptide within the protein scaffold constrains the conformations which the peptide may take. This increases the binding affinity and specificity compared to libraries of free peptides, but reduces the target binding capability of the library. The Affimer protein scaffold was developed initially at the MRC Cancer Cell Unit in Cambridge then across two laboratories at the University of Leeds. [41] [42] [43] [44] Affimer technology has been commercialized and developed by Avacta Life Sciences. X-Aptamers are a new generation of aptamers designed to improve on the binding and versatility of regular DNA/RNA- based aptamers. X-Aptamers are engineered with a combination of natural and chemically-modified DNA or RNA nucleotides. Base modifications allow incorporation of various functional groups/small molecules into X-aptamers, opening a wide range of uses and a higher likelihood of binding success compared to standard aptamers. Thiophosphate backbone modifications at selected positions enhance nuclease stability and binding affinity without sacrificing specificity. [45] [46] X-Aptamers are able to explore new features by utilizing a new selection process. Unlike SELEX, X-Aptamer selection does not rely on multiple repeated rounds of PCR amplification but rather involves a two-step bead-based discovery process. In the primary selection process, combinatorial libraries are created where each bead will carry approximately 10^12 copies of a single sequence. The beads operate as carriers, where the bound sequences will ultimately be detached into solution. In the secondary solution pull-down process, each target will be used to individually pull down the binding sequences from solution. The binding sequences are amplified, sequenced, and analyzed. Sequences that are enriched for each target can then be synthesized and characterized. [47] X-aptamers are commercially produced under the name "Raptamers" by a company called Raptamer Discovery Group. [48] AptaBiD or Aptamer-Facilitated Biomarker Discovery is a technology for biomarker discovery. [49] AptaBiD is based on multi-round generation of an aptamer or a pool of aptamers for differential molecular targets on the cells which facilitates exponential detection of biomarkers. It involves three major stages: (i) differential multi-round selection of aptamers for biomarker of target cells (ii) aptamer-based isolation of biomarkers from target cells and (iii) mass spectrometry identification of biomarkers. The important feature of the AptaBiD technology is that it produces synthetic affinity probes (aptamers) simultaneously with biomarker discovery. In AptaBiD, aptamers are developed for cell surface biomarkers in their native state and conformation. In addition to facilitating biomarker identification, such aptamers can be directly used for cell isolation, cell visualization, and tracking cells in vivo. They can also be used to modulate activities of cell receptors and deliver different agents (e.g., siRNA and drugs) into the cells. Aptamers have also been against several pathogens both bacterial [54] & viruses including influenza A and B viruses, [55] Respiratory syncytial virus (RSV) [55] and SARS coronavirus (SARS-CoV) [55] in various experimental settings. Aptamers have an innate ability to bind to any molecule they're targeted at, including cancer cells and bacteria. Bound to a target, aptamers inhibit its activity. Aptamers suffer from two issues that limit their effectiveness. Firstly, the bonds they form with target molecules are usually too weak to be effective, [ citation needed ] and second, they're easily digested by enzymes. Adding an unnatural base to a standard aptamer can increase its ability bind to target molecules. A second addition in the form of a "mini hairpin DNA" gives the aptamer a stable and compact structure that is resistant to digestion, extending its life from hours to days. [ citation needed ] Aptamers are less likely to provoke undesirable immune responses than antibodies. [ citation needed ] The ability of aptamers to reversibly bind molecules such as proteins has generated increasing interest in using them to facilitate controlled release of therapeutic biomolecules, such as growth factors. This can be accomplished by tuning the affinity strength to passively release the growth factors, [56] along with active release via mechanisms such as hybridization of the aptamer with complementary oligonucleotides [57] or unfolding of the aptamer due to cellular traction forces. [58] Aptamers have been used to create hot start functions in PCR enzymes to prevent non-specific amplification during the setup and initial phases of PCR reactions. [59] The carrier effect in the secondary response to hapten-protein conjugates. I. Measurement of the effect with transferred cells and objections to the local environment hypothesis Until the mid-twentieth century, immunology had been very much a matter of soluble antibodies and their effect on the antigens of bacteria and viruses. Then, in the wartime and post-war years, a new area opened, of cell-mediated immunity, driven initially by interest in the ubiquitous rejection of homografts in man and animals. Experimental tolerance was a key discovery, that introducing donor-type cells before the ability to reject homografts had developed could prevent the rejection. Haᘞk in Czechoslovakia made the discovery independently in 1953 and by Billingham, Brent, and Medawar in Britain in 1954. Proof that rejection of homografts is immunological in nature came from the discovery by the Medawar group that skin grafts are rejected more rapidly if the host has already rejected previous grafts from the same donor. My contribution was to show that this accelerated reaction could be transferred from one inbred mouse to another by means of spleen cells (1), work that I later continued in the laboratory of George Snell at Bar Harbor, ME (2). Returning to UK, and after a period in Edinburgh University, I joined the National Institute of Medical Research, where Medawar had become director. My experience in Edinburgh with chicken erythrocytes had taught me the value of radioactive labeling (3), so I sought to adapt this technology (fairly new at the time) to tracking serum antibody levels in the small blood samples available in mouse studies. Down the passage worked Rosalind Pitt-Rivers, discoverer of the thyroid hormone T3, a great friend. Jointly we designed NIP-CAP, a structure related to T3 that can (i) serve as a powerful hapten because of its nitro and hydroxyl groups, (ii) couple smoothly to proteins to form part of immunogenic molecule, and (iii) can be prepared in radioactive form at the iodine residue and thus be used to assay binding of NI 131 P-CAP to its antibody (4). Together these properties opened the way to an easy mouse serology indeed for a while, it became so widely used that the European Journal of Immunology accepted its name as not requiring further explanation. My work focused on an aspect of immunological memory, the carrier effect. An individual primed by injection of a hapten–protein conjugate makes a full secondary anti-hapten antibody response only to the same conjugate, but not to the same hapten conjugated to another protein. This finding suggested to us that two cells might be involved, one recognizing the hapten and the other the carrier protein. To explore this possibility, we devised a serology applicable in mice (5). The small samples of serum available were appropriately diluted and then incubated with 10 𢄨 M NI 131 P-CAP their immunoglobulin was then precipitated by addition of ammonium sulfate solution and centrifuged, carrying the bound radioactive hapten down with it. By this method, anti-NIP antibody could be detected down to a concentration of 縐 𢄩 M, as available with adoptively transferred spleen cells. This transfer system could then be used to explore the carrier effect as defined above. The secondary response obtained from the transferred spleen cells was indeed much reduced (-fold) when the cells were stimulated with the same hapten (NIP) attached to a different carrier protein such as bovine serum albumin, compared to stimulation with the NIP-chicken γ-globulin originally used to immunize the cell donor. Importantly, the transferred anti-NIP response could be inhibited by injecting an excess of carrier protein, indicating that the carrier protein was itself recognized independently of the hapten that it carried, and thus that a second population of reactive cells was involved independent of those that recognized the hapten. Our experimental design took spleen cells from mice immunized with NIP-OA (NIP conjugated with ovalbumin) plus adjuvant and transferred them into irradiated host mice that were then challenged with either NIP-BSA (NIP conjugated to bovine serum albumin) or NIP-OA (NIP conjugated to ovalbumin), both without adjuvant. The molar concentration anti-NIP antibody made in response was then measured, and its level titrated against the quantity of antigen in the challenge. Typically, mice needed a higher dose of the heterologous antigen (NIP-BSA) than of the homologous one (NIP-OA) to achieve the same level of anti-NIP antibody. Adding spleen cells from mice immunized with BSA alone to the transferred cell population increased sensitivity to NIP-BSA 10-fold, a finding that defines the rrier effect.” The effect is specific, as the increase was not obtained with spleen cells from mice immunized with HSA (human serum albumin). These BSA-primed cells did not contribute directly to the anti-NIP antibody, as judged by allotype markers on the antibody they acted only as “helper cells.” Thus, these findings reveal a carrier effect mediated by the immune system, but not by antibody. To test for a T cell-mediated effect, cells were obtained from the spleen of mice that 7 days earlier had been irradiated and then reconstituted intravenously with 90 × 10 6 syngeneic thymus cells and immunized with BSA, alum, and pertussis (6). These cells were tested for helper activity by transfer into irradiated syngeneic hosts, along with the usual NIP-BSA as immunogen. The transfer significantly increased the host anti-NIP antibody response, in proportion to the number of BSA-primed cells transferred. Such experiments became easier later, when T cells could be manipulated by means of anti-theta antibody [reviewed by Raff (7, 8)]. By then, it had become clear that cooperation between T and B cells as revealed by the 1971 study considered here, most likely works through an antigen bridge between epitope-specific receptors on both cells. B cells, with their immunoglobulin receptors, recognizing the matrix of epitopes presented on the surface of T cells, became and remain the accepted mechanism of T𠄻 cooperation in the immune response. T cells and their interactions with other cells have become a major theme in immunology. Th interactions lie at the heart of inflammation and other aspects of immunological and infectious disease, and are increasingly being manipulated via monoclonal antibodies directed at cell surface markers and via cytokines. Advances in the molecular biology of the cell underpin these developments. These are extremely active fields, with much to offer in molecular cell biology and via therapeutic intervention. The innate and adaptive immune responses discussed thus far comprise the systemic immune system (affecting the whole body), which is distinct from the mucosal immune system. Mucosal immunity is formed by mucosa-associated lymphoid tissue, which functions independently of the systemic immune system, and which has its own innate and adaptive components. Mucosa-associated lymphoid tissue (MALT) , illustrated in Figure 23.15, is a collection of lymphatic tissue that combines with epithelial tissue lining the mucosa throughout the body. This tissue functions as the immune barrier and response in areas of the body with direct contact to the external environment. The systemic and mucosal immune systems use many of the same cell types. Foreign particles that make their way to MALT are taken up by absorptive epithelial cells called M cells and delivered to APCs located directly below the mucosal tissue. M cells function in the transport described, and are located in the Peyer’s patch, a lymphoid nodule. APCs of the mucosal immune system are primarily dendritic cells, with B cells and macrophages having minor roles. Processed antigens displayed on APCs are detected by T cells in the MALT and at various mucosal induction sites, such as the tonsils, adenoids, appendix, or the mesenteric lymph nodes of the intestine. Activated T cells then migrate through the lymphatic system and into the circulatory system to mucosal sites of infection. Figure 23.15. The topology and function of intestinal MALT is shown. Pathogens are taken up by M cells in the intestinal epithelium and excreted into a pocket formed by the inner surface of the cell. The pocket contains antigen-presenting cells such as dendritic cells, which engulf the antigens, then present them with MHC II molecules on the cell surface. The dendritic cells migrate to an underlying tissue called a Peyer’s patch. Antigen-presenting cells, T cells, and B cells aggregate within the Peyer’s patch, forming organized lymphoid follicles. There, some T cells and B cells are activated. Other antigen-loaded dendritic cells migrate through the lymphatic system where they activate B cells, T cells, and plasma cells in the lymph nodes. The activated cells then return to MALT tissue effector sites. IgA and other antibodies are secreted into the intestinal lumen. MALT is a crucial component of a functional immune system because mucosal surfaces, such as the nasal passages, are the first tissues onto which inhaled or ingested pathogens are deposited. The mucosal tissue includes the mouth, pharynx, and esophagus, and the gastrointestinal, respiratory, and urogenital tracts. The immune system has to be regulated to prevent wasteful, unnecessary responses to harmless substances, and more importantly so that it does not attack “self.” The acquired ability to prevent an unnecessary or harmful immune response to a detected foreign substance known not to cause disease is described as immune tolerance . Immune tolerance is crucial for maintaining mucosal homeostasis given the tremendous number of foreign substances (such as food proteins) that APCs of the oral cavity, pharynx, and gastrointestinal mucosa encounter. Immune tolerance is brought about by specialized APCs in the liver, lymph nodes, small intestine, and lung that present harmless antigens to an exceptionally diverse population of regulatory T (Treg) cells , specialized lymphocytes that suppress local inflammation and inhibit the secretion of stimulatory immune factors. The combined result of Treg cells is to prevent immunologic activation and inflammation in undesired tissue compartments and to allow the immune system to focus on pathogens instead. In addition to promoting immune tolerance of harmless antigens, other subsets of Treg cells are involved in the prevention of the autoimmune response , which is an inappropriate immune response to host cells or self-antigens. Another Treg class suppresses immune responses to harmful pathogens after the infection has cleared to minimize host cell damage induced by inflammation and cell lysis. CAROLYN S. FELDKAMP , JOHN L. CAREY , in Immunoassay , 1996 The antigen binding site is formed from the amino-terminal ends (variable domains) of L and H chains. The two chains are folded to form globular variable domains, VH and VL, similar to the folded domains of the constant regions. Looped segments called complementarity defining regions (CDRs) at the ends of the molecule are formed by hypervariable regions which are brought close together in the three-dimensional structure. In individual antibodies, VH and VL form a cleft or trough of somewhat variable size (approx. 2 × 2 × 1 nm deep) as observed by x-ray crystallography, into which the epitope fits and binds with high affinity. The actual points of contact in the few antigen-antibody systems which have been studied are 4–6 to 16–17 amino acid residues, usually charged( 13 ). Deep within the site, hydrophobic residues predominate. As the cleft may be larger than the original epitope, it is clear that any antibody can conceivably bind other epitopes, especially those with similar structure, as long as the charged residues in either the antigen or the antibody in the binding site are compatible (no electrostatic repulsion). Heteroclitic antibodies have been described which have binding sites that can accommodate amino acid changes in antigen causing an increase of several orders of magnitude in binding affinity. There have been detailed studies on only a few antigen-antibody pairs, but the results can be extrapolated to understand cross reactivity and specificity in immunoassays. The hapten will be attached to the carrier molecule, and the hapten should have the epitope. Epitope is required in the hapten. What are the different strategies to come up which involved the hapten design? First strategy was really not the transition state analogue method. The simplest strategy (transition state analogue strategy) was that you know the transition state, make an analogue of transition state, attach it to the protein, and generate the monoclonal antibodies. But that was not the initial studies that were made. This whole catalytic antibody was discovered in the western coast in the California particularly in an institute called Scripps Research Institute, where two scientists Richard Lerner and Peter Schultz developed this technology on catalytic antibody. So, the first examples were based on producing the catalytic site in the antibody, because every enzyme has a catalytic site. This catalytic site contains amino acid residues that help in doing the reaction.. In the catalytic site of chymotrypsin, the catalytic triad is comprised of serine, histidine and aspartate. So, if you can generate them artificially at the proper position, then you can make an antibody which will catalyze like chymotrypsin. But to generate three different amino acids at designed positions is really difficult. So, people went for actually producing only one active site amino acid, which is involved in catalysis,. So, the first technique that was used was basically generating the catalytic site which will catalyze the reaction. I think if I give an example, then it will be much clearer. See there is a molecule: nitro phenyl moiety with fluorine at the benzylic carbon and then there is a β-hydrogen. And this hydrogen is quite acidic because there is a carbonyl group attached to it. We know that if I want to a make double bond here, I will definitely add a base. The mechanism for the reaction is that the base abstracts the hydrogen and the C-H bond electron pair goes here and the fluoride leaves. So, that is the simple mechanism. It is a We have not specified R, because sometimes R may be some group which is very sensitive to base. So, you cannot use base in that case to induce this elimination, but we know that enzymes work at almost neutral pH, so that is another advantage of catalytic antibody instead of base (which can do this elimination), can we can generate a catalytic antibody which will also do this elimination process? That will be very useful because that will be under very mild condition. Of course, this is an example which is nothing but they wanted to have a proof of concept that yes this is possible. You can generate the particular amino acid responsible for catalysis at the catalytic site which is the active site. If I have the antigen which is invading our system, suppose that antigen has lot of amino acids like lysine.. Its surface will be full of lysines which will be positively charged at physiological pH. Now, if an antibody is generated against it, then we can definitely assume that the antibody must be such that it must be able to recognize this antigen. Thus the antibody must be such that it has got lot of carboxylate groups. Because only then there will be an interaction with the NH3 plus, otherwise there will be possibly no recognition between the antigen and the antibody. Thus for fruitful recognition, if the antigen has lot of positive charge on the surface, the antibody will have lot of negative charge on its surface when it binds. Similarly, if the antigen has lot of aromatic rings, then the antibody will also have aromatic rings because that will give you π-π stacking interactions. So, these are some very simple things with which you can predict the probable structure of the antibody depending on the structure of the antigen. Now, in this case remember the antigen what you are using is basically your reaction substrate. So basically I want to generate an antibody which is a protein instead of the base, the protein has a base at that position where the hydrogen is residing. If the protein has a base which is generated here by manipulation, then the same reaction will take place, because what you need is a base in proximity with the hydrogen. This molecule binds to this antibody that is also very important some binding parameter has to be there for which it binds and the base is right in front of the hydrogen. So, now the base will abstract the hydrogen and the elimination process will take place. Now, that means, what you have to do? When you design the hapten, you have to think that how can I generate a basic center at the catalytic site. I already told you that if the antibody is positively charged, then you get a negative carboxylate in the antibody. Now, here you want a basic group. What is a basic group in a protein at biological pH? Sometimes we think that the basic groups are arginine, lysine etc. But the problem with this concept is that at the biological pH, lysine is present as a NH3 plus its lone pair is no longer available similarly arginine will be also present as the positively charged conjugate acid. So, they are not bases in the biological system (at the pH 7.2). So, what are the bases then in biological system? A base is something which has got excess electron, either the lone pair or it could be negatively charged like OH minus. Which amino acid residue exists as the negative charge in biological conditions? The amino acids that have carboxylic side chain because carboxylic acid at biological pH will exist as carboxylate like glutamate or aspartate present as the anion so that is your source of base. So, if that means I have to generate a carboxylate at the active site, how can I do that? See here it is written if I have a hapten like this where I put a NHMe plus here, that means, the hapten has a plus charge somewhere in the middle (where there was the hydrogen attached). So, I have a positive charge here. I keep this aromatic ring intact other things more or less intact only I have to attach the large carrier protein. So, up to this point is your hapten and then you attach it to the protein this is the hapten. In the hapten design, you will notice that this part is not changed. That is because this part will make another aromatic amino acid here which will assist recognition thus this nitro phenyl group has a stabilizing interaction by π- stacking. And this NHMe plus will generate a carboxylate here in order to have this antigen- antibody stabilizing interaction. Exactly that was done. So, this molecule was injected to mice, and the monoclone antibody was isolated. And each had an aromatic group here which stabilizes the nitro phenyl moiety and it had a carboxylic group right where you wanted, and this then underwent elimination reaction as desired. So, this is one of the first examples of catalytic antibody, but again I repeat, that this is not the transition state analogue approach, this is an approach which directs the generation of the catalytic residue in the antibody. Now, let us go to other examples. I just told you that this is the first strategy which involves the generation of catalytic residues. I have given you generation of a carboxylate. Similarly if in some reaction, you want generation of a positive charge (something like NH3 plus), then you have to use a carboxylate in the hapten, which is just the reverse way. The next one is the transition state analogue approach. And the first reaction that was tried was the hydrolysis of an ester. Here this is X suppose this X is OMe we are considering alkaline hydrolysis of an ester. Now, this is also called saponification (alkaline ester hydrolysis). Now, the mechanism of ester hydrolysis by base is discussed here. Suppose this is your base. So, the base first attacks and forms what is called a tetrahedral intermediate this is a tetrahedral intermediate. And this is your rate determining step, this attack by the nucleophile on to the carbonyl carbon. It is a bimolecular reaction, two molecules are involved to form the intermediate via the transition state. And the next step is this O minus comes back and this leaving group leaves. So, you get the hydrolyzed product. This is Y, suppose the Y is OH minus then you get the carboxylic acid. That means, in hydrolysis of an acyl system, an ester goes through a tetrahedral intermediate The intermediate is the one which is closest to the transition state. And so when I do the hapten design, I can take the intermediate and see which molecule resembles this intermediate the intermediate is stable, because if it is not stable, then nobody could isolate this. You have to understand that once it becomes O minus, it is very transitory this comes back and X leaves. So, you cannot have this taken in a test tube and seal it and that will remain forever that is not true, this is also transitory. So, what you need is a stable molecule which looks like this that means you should have a molecule which is because this intermediate is tetrahedral secondly, it should have a negative charge on the outward atoms. So, what you need is your R group I said that do not disturb the R group because depending on the R group, your binding parameters are decided. What you do is take a phosphonate. See basically in phosphonate, phosphorus is directly attached to a carbon . P is attached to O (double bonded), and O minus, OR and R (single bonded). This is a stable molecule. Now, look at the structure of the tetrahedral intermediate and the phosphonate instead of carbon, what you have is a phosphorous, but this is tetrahedral. And this has also got a negative charge like the carbon O minus, you have phosphorous O minus. So, instead of carbon, you have just a phosphorous, which is just slightly bulkier then carbon that is the only difference. Let us take an example. This is A, where this is the ester. You want to hydrolyze it, via normal saponification using OH minus. So, this is the tetrahedral intermediate, and in this tetrahedral intermediate, it comes here, and this group leaves this groups leaves here. So, you get the carboxylic acid and the corresponding phenol. In this case, the phenol is the alcohol part so that is the mechanism. This is your intermediate. So, what will be your hapten? The hapten is this part which you do not change again I repeat that this part remains the same. Now instead of carbon, you take a phosphonate P double bond O, O minus and the other part remaining the same. Only thing is that through this R, you attach it to a linker and then the protein.. So, this is the hapten, it resembles perfectly the intermediate and the intermediate is close to the transition state. So, if you can generate an antibody which recognizes this ensemble, then that is going to catalyze the hydrolysis of this compound so that is very simple, and they Suppose you want to hydrolyze an amide like all those proteases which hydrolyze the amide bond. You can do it with alkali. So, if you add alkali, again you have that tetrahedral intermediate, and then the tetrahedral intermediate collapses, and this goes out, and you get the carboxylate and the nitro amine. You want a catalytic antibody to catalyze this reaction. So, now you generate the hapten. Here instead of carbon, you have nitrogen. So, this is nitrogen, adjacent to nitrogen is this phosphorous. This is called phosphoramidite. So, this is very simple the reactions are not very difficult. The reactions basically involve synthesis of catalytic antibodies for the hydrolysis of amide or for the hydrolysis of ester. They have done also hydrolysis of carbonate. You might say that sir these reactions are quite easy, but as I said in the actual case, you might have certain groups which react under these conditions. This is very mild when you use the catalytic antibody. And the other thing is that, since these are enzyme like reactions, the turnover number will also be very high unlike the hydrolysis under the basic conditions (saponification), the turn over number is also very high. Now, we come to a more difficult reactions. You know that some of the amino acids are essential, some are non-essential amino acids. Essential amino acids mean you have to give them from outside. Some of the microorganisms, can make one particular amino acid like phenylalanine which we cannot make phenylalanine has to come from outside. Now, the biosynthesis of phenylalanine is little complex, but we have taken only some intermediate steps where phenylalanine, biosynthesis is involved. This is a molecule which is called chorismic acid. There is some biosynthetic path way through which this molecule is An enzyme which plays a major role is this biosynthesis is chrosmic mutase. But actually, it is chorismate, because all these acids are actually present in the carboxylate form that is how you always have glutamate, aspartate. So, this will be chorismate mutase it is known as mutase because basically it is an isomerization reaction where the skeleton changes isomerization means molecular formula of these is same as that. So, it must be a kind of a So, that isomerization reaction is done by chorismate mutase mutase means where the skeleton changes. The skeleton looks entirely different from what was there. Now, if you want to do this reaction chemically, you can do itbut you have to heat it at a very high temperature. This is an example of sigmatropic reaction. Specifically this is called a [3,3] sigmatropic reaction. In these, both ends of the relocating σ bond migrate three atoms. If you look at all these, the sigma bond that is broken is this one and then this will have the numbering as shown. So, this becomes 3 and this is also 1, and that is 2 and that is 3. So, ultimately the 3 and 3 carbons are combining with each other, so that is why called this is called a [3,3] sigmatropic shift. This is not the actual geometry through which the reaction takes place. The actual geometry is shown here. It takes the shape of something like an inverted boat. And then you have this oxygen, you have that carbon, then the double bond, and this CO2 minus, and here is double During the reaction, the molecule takes up this geometry. You may argue that the carbon atoms present the 3, 3 positions are quite far apart, how are they reacting with each other? But in this structure (that has been shown), you can there is no such problem. So, this is the O then that double bond and this is the CO2 minus, and here you have a CO2 minus. So, when the reaction takes place, the arrow goes like this, you have a transition state which will looks So, this is the your this is the oxygen, this is the double bond, this is the CO2 minus, and earlier there was a double bond here and a double bond there, and there was OH here. So, now, the reaction goes by this pathway as shown. So, what you have is oxygen, then you have this which id close to a chair. If you look at this position, more or less like a chair. So, it is a chair like transition state which is involved in this process, and that is true for all [3,3] sigmatropic reactions they go via chair like transition state. Examples are Cope rearrangement, Claisen rearrangement etc. Now, earlier people used to think that pericyclic reactions occur either by a heat or by light. People used to believe that pericyclic reactions cannot be catalyzed. They are unaffected by catalysts. But here is an enzyme chorismate mutase which does the rearrangement very fast at room temperature you do not have to heat it. So, that actually broke the myth that pericyclic reactions can be catalyzed. How the enzymes are catalyzing it? Basically the enzyme helps the molecule to adopt a conformation through which the reaction takes place. The enzyme helps the molecule to bind, adopt a conformation through which the reaction takes place. So, if you want to make a catalytic antibody to have this reaction catalyzed, what you need to do is make a transition state like molecule which is stable like this. See this is the transition state which we are talking about there is a OH here earlier remember there is a OH here in chorismic acid, so via that OH, you attach it to the to the linker. The linker was a diazo compound and through the diazo compound, you attach the protein because diazonium salts are prone to attack and the nitrogen leaves. So, by that you can attach your protein part. So, basically what you have done? You have made a molecule which looks like the transition state, but this is stable you synthesize this molecule and via this OH, you attach it to the carrier protein now you generate catalytic antibody and that antibody has been very successful that antibody catalyzed this chorismate mutase reaction. Chorismic acid going to prephenic acid. This is the intermediate for the phenylalanine synthesis so this is a very critical step. Since this is an essential amino acid, so this has to come from outside. The question is how the plants or the microorganisms make phenylalanine? So, this is the route. So, chorismic acid rearranges to the prephenic acid, then there is a decarboxylative dehydration but here OH is a bad leaving group, so is converted to a phosphate, so that leaves and that gives you a compound which contains this CO CO2H ( α-keto acids). When we will read some more coenzyme chemistry, we will see that these α-keto acids can be converted to amino acids. So that is the story of phenylalanine and that has come because of The last reaction that I am going to discuss is involving those reactions which are disfavored reactions which do not happen under standard organic reaction conditions. Here you can force the reaction by catalytic antibody to follow a disfavored path way. How does this happenLet us take this example, this is a phenyl with some substituent, then CH2CH2 and an epoxide and then the epoxide is connected ultimately to a hydroxyl group. Now, if you add some acid, this the epoxide will be protonated and as soon as its protonated, since epoxides are strained molecule, they will open up. Their opening is assisted by this adjacent nucleophile which is OH. So, now you have two modes of reopening where by the OH can attack either of the two carbons involved in the three membered epoxide ring. One is called 5-exo-tet. What is tet? Tet means you are attacking at a tetrahedral carbon, because these are tetrahedral carbon sp3 hybridized, so tet. Why it is called 5-exo? That means, when it is attacking that one, what you are making is a 5 member ring, so that is 5. And your whole epoxide is outside the ring that is being formed, so that is why that is called The other possibility where the lone pair attacks this carbon, now this whole bond containing the epoxide becomes a part of the ring, so this will be called endo, but again you are attacking to a tetrahedral carbon, but you are making a 6 member ring, so this is called 6-endo-tet. There are some rules called Baldwin’s rules (proposed by Sir Jack E. Baldwin) which govern these cyclization reactions. If you do this reaction, you will see that this is the product which is formed because Baldwin’s rules say that 5-exo-tet is favored and the 6-endo-tet is So, in the in the test tube, if you do this reaction and the chemical reagent used is acid, like some Lewis acid, you will get this product as the major product but if your target is to make the other possible compound, then the question is how to make a disfavored reaction favored? So, what was done is that when this OH attacking in the disfavored reaction which is the part of this C-O bond is now broken so this will be δ- and this oxygen will be δ+ maybe I can draw the transition state so that is δ- and then what you have is oxygen these are half bonds ( these are not formed fully) and this is the aromatic ring and that will be δ+. Now if you can mimic this with a molecule then that will be your hapten, and with that hapten, if you can generate a catalytic antibody and give it to this system, then that will do this reaction (6- So finally they come out with a molecule which is an N-oxide molecule. Now, look at this molecule and try to check here, see what you need is a carbon here which may not be carbon, you can have different atoms, but it should be attached to a kind of negatively charged oxygen. Instead of the carbon you a have nitrogen here and you have O minus here. But what you needed is a δ+ center at the next carbon and you want a minus on oxygen. Now, because this nitrogen is positively charged N-oxide so due to inductive effect, this will generate a δ+ on the adjacent carbon as shown. So, now if you look at this one and that one, they are they resemble each other. See this is tetrahedral, that is tetrahedral, this has got a negative charge, this is the negative charge and this is the positive charge and this positive charge is generated by inductive effect and this R is utilized to attach the protein. So, now if you can generate a catalytic antibody against this, then that will act as a catalyst do a disfavored reaction and ultimately you get this product. In fact, this was done and this was a very classic example and it showed the power of catalytic antibody to do unfavored reactions. There are many more examples of unfavored reactions by using catalytic antibody approach. We do not know any other approach by which you can get a disfavored reaction into a favored process. So, I think that ends this aspect of synthetic biology that involves the Antigen is any substance or molecule that can trigger an immune response in an animal including humans. An antigen is basically anything that is foreign to the body and which can react specifically with an antibody. An understanding of the basic characteristics of antigens and/or pathogens that spark immunological responses in the body is vital for us to know how antigen-antibody reaction actually occurs. It also helps us to know what this complex reaction (i.e., antigen-antibody reaction) implies in clinical terms. Antigens consist of microorganisms such as pathogenic viruses, bacteria, fungi and parasites or worms and helminthes. They may also include proteins, glycolipids, polysaccharides, and harmful chemicals, substances or molecules released by any of these pathogens, and which the immune system of the host organism(s) considers to be non-self molecules. In transplantation immunology where graft or organs are being transplanted from one host to another, the recipient immune system (especially in the case of non-identical twins) can sometime see the transplanted tissues or cells as antigens because they may contain some markers which the non-self receiver’s immunological makeup sees as ‘non-self’ molecules. However, not all foreign molecules that enter the body are capable of sparking up an immunological response that leads to antibody production. Antigens that induce the production of antibodies by the immune system are generally called immunogens. All immunogenic substances are always antigenic because they are able to react and be recognized by a specific antibody. However, some antigens (especially those with low molecular weight) are not immunogenic in nature even though they may exhibit some features of an antigen and are said to be antigenic. Substances with low molecular weight and which are not immunogenic by themselves but can become immunogenic when coupled to a carrier molecule such as a protein (which is immunogenic) are generally called haptens.Drugscan sometimes become immunogens when they spark up allergic reactions in the host taking them. Many biologically and chemically important substances such as drugs, steroid hormones, and peptide hormones can also serve as haptens. Dinitrophenol (DNP), an organic compound is a typical example of a hapten. The phrases antigen and immunogens are often used interchangeably. Immunogenicity is the ability of a substance to elicit an immune response (both humoural and cell-mediated immunity), and this phenomenon is usually exhibited by immunogens. Antigenicity on the other hand, is the property of an antigen that allows it to react specifically with the product of the specific immune response (i.e., antibodies or receptors of T cells). Though related and often confused in immunological discussions, immunogenicity and antigenicity are two distinctive terms with varying immunological properties and functions as it relates to antigens. In immunological terms, antigens are generally referred to ‘substances that can react specifically with the antibody receptor of B cells or T cell receptor when complexed or joined with major histocompatibility complex (MHC) molecules. Further reading William E.P (2003). Fundamental Immunology. 5 th edition. Lippincott Williams and Wilkins Publishers, USA. Stevens, Christine Dorresteyn (2010). Clinical immunology and serology. Third edition. F.A. Davis Company, Philadelphia. Silverstein A.M (1999). The history of immunology. In Paul, WE (ed): Fundamental Immunology, 4 th edition. Lippincott Williams and Wilkins, Philadelphia, USA. Paul W.E (2014). Fundamental Immunology. Seventh edition. Lippincott Williams and Wilkins, USA. Male D, Brostoff J, Roth D.B and Roitt I (2014). Immunology. Eight edition. Elsevier Saunders, USA. Levinson W (2010). Review of Medical Microbiology and Immunology. Twelfth edition. The McGraw-Hill Companies, USA.Split aptamers Edit
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The discovery of T cell𠄻 cell cooperation
by Mitchison NA. Eur J Immunol (1971) 1:10𠄷. doi:10.1002/eji.1830010204
Mucosal Surfaces and Immune Tolerance
IMMUNE FUNCTION AND ANTIBODY STRUCTURE
3.2. Antigen Binding Site
Module 1 : Synthetic Biology and Central Dogma
ANTIGEN
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