Antigen (Ag) (literally standing for "antibody generator") is every molecule that binds at least 1 type of receptor in adaptive immune system.They can be classified according to their ...

• ...... origin
• self
• non self
• ... capability to rise an immune response (immunogenicity)
• immunogen : nonself molecules degradable and processable by APCs, with M_r > 5 kDa (100 kDa is the optimal) and physicochemical complexity. They may be further classified as follows :
• natural
• artificial (in vitro-obtained fragment of a natural protein)
• synthetic
Regarding humoral immune responses immunogens are divided into 2 groups :
• thymus-dependent (TD) Ags : B lymphocytes need IL-2 from activated T lymphocytes to become plasma cells
• thymus-independent (TI) Ags : large, multivalent molecules that directly induce B lymphocyte activation to plasma cells (i.e. to antibodies production) without T cell help. Neither memory cells nor class switch occur (=> only IgM and IgG₂ are produced and no affinity maturation occurs) : long-term protective vaccination against them is possible only if they are conjugated to a T-dependent carrier. Anyway short-term protective vaccination against extracellular mitogens is possible even in individuals with T-cells immunodeficiencies.
• type I T-independent Ags (TI-1) (at high concentrations they act as B cell mitogens, while at low concentrations they only induce antibody production in BcR-specific B lymphocytes).
• HBcAg from HBV
• Staphylococcus aureus protein A (SpA) binds to both soluble and membrane bound C_H3 domains of IgG. It is a B cell superantigen that forms a complex with lymphocytes expressing BcRs with clan-V_HIII-encoded variable regions. These BcRs are displayed by 5-10% of mature mouse B cells and 50% of human B cells, including marginal-zone and follicular B cells, and B1 cells with a BcR antigen-binding region of the T15 idiotype. In vitro, the initial response of B cells to SpA is similar to the normal response to antigen exposure. After 2 hours, levels of the targeted cell-surface BcR are decreased; after 16 hours, the BcR co-receptors CD19 and CD21 are downregulated, and the activation markers MHC class II, CD69 and CD86 are upregulated. After 48 hours, despite certain B cells having undergone 2-3 rounds of proliferation, their overall number is decreased by 36%, indicating increased rate of activation-induced cell death (AICD)
• Moraxella catarrhalisIgD binding protein (MID) binds to C_H1 domain of mIgDs.
• BcR-independent : B cell proliferation and polyclonal antibody production in response to Gram-negative bacterial infection are modulated by acyloxyacyl hydrolase (AOAH), a host enzyme that deacylates bacterial LPSs. Deacylation of LPS occurred over several days, allowing LPS to act as an innate immune stimulant yet limiting the eventual amount of B cell proliferation and polyclonal antibody production. Control of LPS activation by AOAH indicates that mammals can regulate immune responses to bacterial infection by chemical modification of a TLR agonist^ref
• TLR4 agonists
• TLR7 agonists
• TLR9 agonists
• TLR10 agonists
• type II T-independent Ags (TI-2) (repetitive epitopes multivalently cross-link BcRs inducing antibody secretion but not cell proliferaion; although not required, T_h cell cytokines enhance the response)
• flagellin
• Ficoll
• 2,4-dinitrophenyl-Ficoll (DNP-Ficoll)
• large (M_r > 80 kDa) capsular polysaccharides with negatively charged groups or no charged group at all
• dextran
• bacterial levan (BL)
• type III O polysaccharide from Streptococcus pneumoniae R36A
• glucuronoxylomannan (GXM) from Cryptococcus neoformans
They are internalized and localized in endosomes and lysosomes of APCs and inhibit T cell responses to peptides. On the contrary zwitterionic polysaccharides (ZPS) isolated from strains of Bacteroides fragilis, Staphylococcus aureus, and Streptococcus pneumoniae type 1 are presented by HLA-DR on APCs and can activate CD4⁺ T lymphocytes : the proliferative response depends on free amino (positively charged) and carboxyl or phosphate groups (negatively charged) that are part of the repeating unit structure.
Ag immunogenicity may be improved by non-immunogenic adjuvants.
• holoantigen : complete antigen, as opposed to hapten.
• hapten : usually M_r < 6 kDa and hence usually monovalent. E.g. :
• dinitrophenyl (DNP)
• (4-hydroxy-3-nitrophenyl)acetyl (NP)
• 2,4-dinitrofluorobenzene (DNFB) induces T_h1 reaction
• fluorescein isothiocyanate (FITC) induces T_h2 reaction^{ref1, ref2}
It may become immunogenic only if ...
• conjugated to an immunogenic carrier (Schlepper) . The carrier facilitates the production of antibodies in the following ways :
• the increased size of peptide-carrier complex is sufficiently large to be recognized and engulfed by APCs
• the protein carrier molecules contain sequences that are T_h epitopes that stimulate the proliferation of T_h. Interaction of T_h with B-cells is essential for a strong immune response.
When an animal is immunized with a peptide/carrier conjugate, Abs are also generated to the carrier protein if this is non-self. Most commonly used immunogenic carriers are :
• keyhole limpet (Megathura cremulata) hemocyanin (KLH). It is an extremely large (M_r = 5 MDa), multi-subunit protein that contains chelated Cu of non-heme origin for O₂-binding : it exists in 2 immunologically and physiologically distinct isoforms, KLH1 and KLH2. Both are present in the hemolymph (pH 7.4) as cylindrical didecarmers (typical for gastropod hemocyanins) consisting of 5 subunits (M_r = 450 kDa) that exist in 5 different aggregate states in Tris buffer pH 7.4, which readily dissociate with moderated pH change : in highly alkaline (pH 8.9) or acidic environments, KLH dissociates into sub-units exposing further epitopes. Each isoform contains 8 functional units (FUs) termed a to h from the N- to the C-terminus. Related proteins exist in the closely related species Haliotis tuberculata (HtH1 and HtH2) and Concholepas concholepas (M_r = 6 MDa). Each KLH mole has :
• > 2,000 amines from Lys
• > 700 sulfhydryls from Cys
• > 1,900 sulfhydryls from Tyr
available for conjugation with hapten molecules. It is usually used as the carrier for the immunization of the animals. KLH may also be obtained from maricultures (mcKLH). KLH is poorly soluble in water, however, which can make it difficult to handle. KLH has no homology to vertebrate proteins.
• bovine serum albumin (BSA) from Bos taurus. It is smaller than KLH (M_r = 67 kDa), so more water-soluble and then usually used for the ELISA determination of Ab titers. As it is a popular protein used in immunoassays to block non-specific binding sites, it should not be used as carrier if future assays involve BSA (for instance as blocking agent)
• ovalbumin (OVA) (M_r = 45 kDa; pI = 4.6) from Gallus gallus is a glycoprotein and the major protein of egg white: produces fewer cross-reacting Ab when used to immunize chickens. OVA_257-264 (LEQLESIINFEKLTEWTS) binds H-2K^b
• bovine (Bos taurus) gamma globulin (BGG)
• chicken (Gallus gallus) gamma globulin (CGG)
• goat (Capra hircus) serum albumin : produces fewer cross-reacting Ab when used to immunize goats
• rabbit (Oryctolagus cuniculus) serum albumin : produces fewer cross-reacting Ab when used to immunize rabbits
• b-lactoglobulin
• diphteria toxin
• tetanus toxin
• thyroglobulin (THY)
• HBV_c128-140 (TPPAYRPPNAPILAAFLPATLTMV)
• multiple antigenic peptides (MAPs) are composed of multiple copies of a single epitope attached to a small, non-immunogenic polylysine core, eg :


MAP resins typically have 4 or 8 peptide arms (branching out from the a- and e-NH₂ groups of the polyLys core matrix), designed to reach the 10 kDa M_r desirable for an optimal immunogen. As the MAP grows, increasing coupling and deprotection times may be needed to compensate for steric hindrance effects in such a large compound. The MAP design maximizes the antigen concentration, with synthesized peptide-antigen accounting for up to 95% of the total weight of the final product. MAPs can be analyzed by reverse-phase HPLC, capillary electrophoresis, peptide sequencing, amino acid analysis, or mass spectrometry. Because it introduces multiple copies of the peptide antigen, the MAP-immunogen produces higher antibody titers. The resulting antibodies are also more specific than those raised against peptide/conjugates, since the MAP-immunogen contains only one antigenic sequence chosen or predicted from the protein sequence. MAP peptides are suitable for direct injection (with adjuvant) for antibody production. Since the peptide is linked through the C-terminus, the MAP techniques favors N-terminal or internal peptides and is not recommended for peptides from the extreme C-terminus. The carrier attachment may also cause some steric hindrance. Use KLH conjugates for C-terminal peptides.
• plant viral proteins
• potato virus X coat protein^ref
If studying invertebrate species, you should use carrier proteins such as KLH and ovalbumin.
To measure the specific anti-peptide response, couple the peptide to a carrier protein (C2, usually BSA or OVA) different from those used for immunization (C1, usually KLH) and perform an ELISA assay. Immunized serum is serially diluted in the C1/peptide-coated microwell plates. The dilution factor at which no anti-peptide antibody binding can be observed is the antibody titer.
• assembled into a multiple antigenic peptide (MAP)
• tolerogen => immune tolerance
The purity of a crude peptide typically decreases as the length increase. Purity > 70% is sufficient for generating or testing antibodies. However, purities greater than 95% are often required for biological activity studies. Typically, peptides of 10-15 residues are often required for biological activity studies. Typically, peptides of 10-15 residues in length are used for raising antisera and the yield of peptide for sequences < 15 residues is often sufficient
• scale 20 => 20 mg crude yield => 10-12 mg purified yield
• scale 50 => 50 mg crude yield => 20-25 mg purified yield
• scale 100 => 100 mg crude yield => 50-51 mg purified yield
Peptides containing a large percentage of hydrophobic residues, such as Leu, Val, Ile, Met, Phe, and Trp, will often have limited solubility in aqueous solution and may be completely insoluble. To help ensure solubility, the hydrophobic amino acid content should be below 50% and there should be at least one charged residue (e.g. Asp, Glu, Lys, and Arg) for every 5 amino acids. Cys, Met, or Trp residues are often problematic in synthesis because these residues are susceptible to oxidation and/or side reactions. If possible, choose sequences that contain a minimum of these residues. Alternatively, conservative replacements can be made for some residues. Peptide solubility is dependent on the amino acid content of the peptide. Test a small amount of peptide for solubility, in water, PBS or various solvents prior to dissolving the entire peptide amount. For most peptides, solubilization in PBS or PBS-azide is used. Depending on the amino acid content, the use of mild acids or bases may be required (e.g. ammonia or acetic acid). If the peptide is not water soluble, employ one of the suggestion below :
• for peptides that are acidic - due to the presence of aspartic and/or glutamic acid residues - add a small amount of 5% ammonium hydroxide
• for peptides that are basic - due to the presence of histidine, lysine, arginine residues - add a small amount of 5% acetic acid. Avoid basic conditions when reconstituting peptides that contain cysteine
• for peptides that are hydrophobic - due to the presence of isoleucine, leucine, phenylalanine, and/or valine residues, dissolve the peptide in a minimal amount of acetonitrile, isopropyl alcohol, ethanol, DMF or DMSO in addition to PBS or as a substitute
Storage :
• most peptides are stable at -20°C indefinitely, especially if they are lyophilized and stored in a dessicator
• allow lyophilized peptides to come to room temperature before exposing them to air. This will minimize moisture-related effects. When lyophilization is not possible, the next best method of storage is in small, working size aliquots at -20°C or -80°C

Model antigens :

• hen egg lysozyme (HEL) : the following epitopes are presented by APCs expressing MHC class II I-A^k molecules after processing :
• epitope 18-33
• epitope 31-47
• epitope 48-63 (50% of responding T cells require Trp at positions 62 and 63)
• epitope 115-129
Minimal epitope required for I-A^k binding : epitope 52-61
Tryptic fragment = fragment 46-61
• ovalbumin (OVA) : epitopes :
• OVA_323-339

They are made up of ...

• epitopic portions or antigenic determinants
• epitopes (usually multivalent : it is said monodeterminant if all epitopes are identical, otherwise multideterminant)
• conformational (lost after denaturation)
• linear or sequential
• always accessible
• accessible only after denaturation
• post-proteolysis or deglycosylation
A large complex protein antigen will contain many possible epitopes which can be classified as
• immunodominant antigenic determinants are those in a complex mixture (such as a whole virus or tumour cell) which elicit the greater T lymphocyte response. Immunodominance (ID) is an issue for vaccine development : it relates to ...
• ability to interact with the MHC molecules of a given individual (chemical dominance), which in turn is due to ...
• sequence selectivity of the proteasomes
• sequence selectivity of the TAP complex
• competition among the peptides for binding to the MHC molecule
• presence of putative holes in the repertoire that would lead to no or low recognition of certain class I complexes
Anyway under inflammatory enviroments (such as protein administered in CFA) mechanisms which are yet unidentified equalize large differences in the levels of peptide presentation, allowing for the generation of T cell response to high- and low-density epitopes and superseding any direct relationship between immunodominance and chemical dominance.
• lymphokines (in particular IL-12) at the site of T cells activation that allows expansion of all T cell repertoires.
• subdominant antigenic determinants
• cryptic antigenic determinants : an antigenic peptide that is generatede at sub-threshold levels. When cryptic epitopes become visible to the immune system they can elicit an immune response that is responsible for autoimmune disease
A mimotope is a compound that mimics the structure of an epitope and provokes an identical
antibody response, useful in the development of vaccines.
If the Ag is a BcR / Ig / Ab, the epitope is referred as idiotype (= V region) and it is made up of 6 idiotopes (= CDR/HVs, 3 per V_H region and 3 per V_L region).
Anti-idiotypic network occurs only in vitro (in vivo only upon artificial induction : e.g. in lymphoma therapy) : FR are conserved and so immunoglobulins are poorly immunogenic.
• agretope (in MHC co-presented Ags) is the portion that binds to MHC molecules. Epitopes recognized by T cells are often internal and contain amphipathic peptide sequences : it is believed that the hydrophobic residues act as agretopes while the hydrophilic residues act as epitopes.
• non epitopic portions

Normally 1 particular TcR is expressed in one T-cell every 10⁵ : as there are 10⁹ T-cells in the human body, only up to 10⁴ T-cells (0.001 %) can be activated by a given epitope. Some exception exist :

• superantigens (SAg) : they bind (out of the peptide-binding cleft) to many kinds (but not all) of HLA-DR b chain on surface of APC (MHC class II^hi, CD40^hi80^lo86^mi205^hi), eventually inducing homodimerization. Then each SAg can bind up to 5 different Vb allotypes of TcR b chain, independently from the associated D, J and Va sequences. The combination of both mechanisms allows activation and rapid proliferation of up to 20% of all T_h-cell clones, leading to a massive release of both lymphocyte-derived (IL-2, TNF-b, IFN-g => activation of macrophages, depression of B lymphocytes => no humoral immunity to further challenges) and monocyte-derived (IL-1b, IL-6, TNF-a) pro-inflammatory cytokines : expansion occurs for 3-4 days, then these activated T lymphocytes are deleted (except that CD4⁺25⁺ regulatory T cells) by Fas-FasL interaction-dependent, activation-induced cell death (AICD) => immunodepression. The remaining T cells expressing SAg-reactive TcR undergo RAG activation and TcR a locus secondary rearrangement => tolerance induction. After continuous exposure, only a limited fraction undergoes protracted expansion and immunogical memory. Examples of superantigenic toxins (SAGTs) :
• requiring His⁸¹ on HLA-DR b chain for Zn coordination :
• inducing T_h1 polarization
• homologous pyrogenic toxins (PTSAgs)
• streptococcal superantigens (SSA) : M Ag, SPE-A, SPE-C and SPE-X / SMEZ from Streptococcus pyogenes
• SEA/H from Staphylococcus aureus (SEB also acts via TLR4)
• YPM from Yersinia pestis
• viral superantigens
• HHV-4 / EBV
• HHV-5 / CMV
• mouse mammary tumor virus (MMTV) superantigens. The C-terminal 30 to 40 amino acids of the superantigen of different MMTVs display high sequence variability that correlates with the recognition of particular TcR Vb chains.
• Mtv29
• I-E-dependent MMTV(GR)
• MMTV(SIM)
• mtv-8
• I-E-independent mtv-7
• inducing T_h2 polarization
• rabies virus nucleocapsid (NC) protein for human Vb8- and murine Vb6-containing TcRs.
• not requiring His⁸¹ on HLA-DR b chain :
• Mycoplasma arthritidis mitogen (MAM) / superantigen (MAS)
• E2-spike protein of SARS coronavirus
• mitogens are large (usually non-peptide) biopolymers made up of many identical epitopes that induce BcR and/or TcR cross-links (patching and capping) => direct activation of B-lymphocytes and/or T-lymphocytes of many antigenic specificities(polyclonal activators), in contrast to an antigen, which only activates cells specific for the antigen => cell division. One can demonstrate mitogen activity by incubating lymphocytes in culture with the mitogen for 48 hours, then adding [³H]-thymidine and further incubating the culture for 5 hours. The cells are now harvested and the incorporated radioactivity determined with a scintillation counter. Control cells (no mitogen) should have cpm < 300, whereas those with mitogen will have cpm >10,000.
• for all T lymphocytes only :
• vegetal lectins
• phytohemagglutinin (PHA) from Phaseolus vulgaris (red kidney bean)
• concanavalin A (Con-A) from Canavalia ensiformis (Jack bean)
• anti-CD mAbs
• anti-CD2 mAbs
• anti-CD3deg mAbs
• anti-CD28 mAbs
• mycolic acids (some are effective only on CD4^-8^- cells, others are effective only on CD4⁺ lymphocytes)
• for both all B and all T lymphocytes :
• pokeweed mitogen (PWM) from Phytolacca americana (a.k.a. Virginian or common pokeweed) consists of pokeweed lectin B (PL-B) : less effective on T cells
• for Ag-specific B lymphocytes only :
• high concentrations of type I T-independent Ags (TI-1)
• because of polyclonal B cell activation, LCMV-specific CD4⁺ T cell help impairs the induction of neutralizing antibody responses. Experimental reduction in CD4⁺ T cell help in vivo resulted in potent neutralizing antibody responses without impairment of CD8⁺ T cell activity^ref.

---

Copyright © 2001-2014 Daniele Focosi. All rights reserved | Terms of use | Legal notices
 About this site  | Acknowledgements  |    |    | Current link partners
Abbreviations and acronyms  |  Medical terminology  |  Add a link  | Translate   | Softwares

---

---

Search PubMedProteinNucleotideStructureGenomeLocusLinkPopSetOMIMTaxonomyBooksProbeSet3D DomainsUniSTSDomainsSNPJournalsUniGene for

Search AboutAltaVistaAsk JeevesDictionaryDirectHitExciteFindWhatGoogleGoogle GroupsHotBotLookSmartLycosMetacrawlerMetaLocateNorthern LightOpen DirectorySnapThesaurusWebcrawlerYahoo for

Search Medical Dictionary  for