The immune system & immunoglobulins Flashcards
Immune response
=> Features a Specialized Array of Cells and Proteins
physiological level: the response of the immune system to an invader–> intricate, coordinated set of interactions among many classes of proteins, molecules, and cell types
level of individual proteins: acutely sensitive and specific biochemical system built upon the reversible binding of ligands to proteins (antibodies) (interações prot.-ligando sem grupo prostético! Numa det. cavidade c/alinhamento das cad. lat.–> max. interação–> orientação 3D altamente específica=> seletividade)
2 complementary immune systems
Cellular immune system
- targets own cells that have been infected
- clears up virus and infecting bacteria
- key players: macrophages, killer T cells (Tc) and
inflammatory T cells (TH1)
(t- thymus)
Humoral immune system (“humor” latin= fluid)
- targets extracellular pathogens (“found in the body fluids”…)
- can also recognize foreign proteins
- makes soluble antibodies
- keeps “memory” of past infections
- key players: B-lymphocytes and helper T-cells (TH2)
(b- bone marrow)
Anticorpos/ imunoglobulinas
• Proteins at the heart of the humoral immune system
• Produced by B lymphocytes (linfócitos tipo B), or B cells, make up of 20% of blood protein
• Bind bacteria, viruses, or large molecules identified as foreign and target them for destruction
“âncora que reconhece virus, pega nele e leva-o ao macrófago…”
1 antibody= 2 identical antigen-binding sites
[extraordinary BINDING AFFINITY AND SPECIFICITY]
[humans capable of producing 10^8 different antibodies w/ distinct binding specificities bc: random reassembly of a set of ig gene segments through genetic recombination mechanism]s
Antigénios
Substances that stimulate production of antibodies
• typically macromolecular in nature (< 5 kDa are generally not antigenic [mas: small mol. covalently attached to large prot. in the lab= haptens can be used…])
• recognized as foreign by the immune system
• coat proteins of bacteria and viruses
• surface carbohydrates of cells or viruses
[Antigen may be virus, bacterial cell wall, individual protein, other macromolecules]
Antibodies specifically bind to antigens!
• Binding will mark the antigen for destruction or interfere with its function.
• A given antibody will bind to a small region (epitope) of the antigen no próprio paratopo
• One antigen can have several
epitopes
Polyclonal and monoclonal antibodies
- Polyclonal antibodies are heterogeneous mixtures of antibodies, each specific for one of the various epitopes on an antigen –> mais específico!! reconhece vários epitopos
- Monoclonal antibodies are all identical, produced by clones of a single antibody-producing cell. They recognize one specific epitope
Cellular immune system
- Antibodies bind to fragments displayed on the surface of invading cells (Ig associam-se a epitopos à superfície do virus–> sinal para endocitose)
- Macrophages: large phagocytes that ingest bacteria that are tagged by antibodies => interação prot.-prot: à superfície, reconhece parte do anticorpo… => reorganização da membrana…=> interiorização
Structure of antibodies
1972, Gerald Edelman and Rodney Porter
3 domínios distintos (“Y”)
two heavy chains and two light chains composed of constant domains (com estrutura caraterística: ig fold!) and variable domains:
• Light chains (“de fora”): one constant and one variable domain (“na ponta”)
• Heavy chains(“dentro, compridas”): three constant and one variable domain (“na ponta”)
[as 4 chains ligadas por lig. não-covalentes e dissulfureto]
Variable domains of each chain make up the antigen-binding site (two per antibody) and some residues are hypervariable, which confers antigen specificity! variação de AA… influencia a associação sem alterar a estrutura.
Também zonas rel. desordenadas (sem estrut. 2ª, c/ algumas pontes dissulfureto)–> movimento! asseguram flexibilidade conformacional –> melhor ajuste…
conformational flexibility is important to the function of ig
Antigen– antibody interactions
Antigens bind via Induced Fit (encaixe-induzido) [ligando--> alteração conformacional (reorganização... para acomodar ligando --> estabilidade, seletividade)]
[Specificity conferred by chemical complementarity in therms of shape+ location of charged, hydrogen-bonding groups; complementarity is achieved interactively as they influence each other as they come together–> induced fit]
A protein antigen (ex lysozyme), binds to the end of a Fab (antigen-binding fragment) domain of an antibody;
The end of the antibody and the antigen have complementary shapes! –>
a large amount of surface can be buried on binding
–> docking muito forte, específico, afinidade alta sempre!
Types of immunoglobulins
ver folhas
- In many vertebrates, IgG is but one of five classes of immunoglobulins.
- Each class has a characteristic type of heavy chain, for IgA , IgD, IgE, IgG, and IgM, respectively.
- Two types of light chain occur in all classes of immunoglobulins.
overall IgD, IgE (associada à resposta alérgica) similar to IgG
IgM (“estrela”) occurs either in a monomeric, membrane bound form or in a secreted form that is a cross-linked pentamer (dissulfide bond, joining chain)
IgA, found principally in secretions such as saliva, tears, and milk, can be a monomer, dimer, or trimer (joining chain, secretory protein, unida topo a topo, envolvida nos fluidos)
Antibody techniques: ELISA
(enzyme-linked immunosorbent assay)
specific reaction of an antibody with its antigen –> basis of several techniques that identify and quantify a specific protein (reta calibração) in a complex sample –> feito em placa (96-well polystyrene plate), extrato celular com n proteínas adsorvem
Procedure (ex):
-coat wells with an HSV (herpes simplex virus) antigen
(-next: block unoccupied sites with unspecific protein)
-incubate with primary antibody against specific antigen, protein of interest (–> antibodies against HSV will bind)
-incubate with secondary antibody-enzyme complex that binds primary antibody (anti– human IgG linked to horseradish peroxidase (enzyme that catalyses colour-forming reaction)
-add substrate of the antibody-linked enzyme
–> formation of coloured product indicates presence of specific antigen+ color intensity proportional to the []
(here greater amounts of HSV antibody turn brighter yellow)
Antibody techniques: western blotting
In western blotting/ immuonoblotting, proteins are:
1. separated in an SDS-PAGE gel (by gel-electrophoresis), transferred to a polymer (nitrocellulose membrane)
- stained with an antibody specific for the protein= primary antibody.
- The secondary antibody (specific for the first antibody) is added.
- The secondary antibody is attached to an enzyme that generates a chemiluminescent product/ contains a fluorescently labeled tag,
- This allows detection of the primary antibody.
Using antibodies to label proteins in cells
Fluorescently labeled antibodies to particular proteins + fluorescence microscopy=> cellular location of proteins to be determined and to follow their movements in the cell = resolução espácio-temporal
–> Fluorescence micrograph of a developing Drosophila embryo (was stained with a fluorescence labeled monoclonal antibody for the DNA binding protein)
[outros modelos bioquímicos: ratinhos- recriar patologias, peixe-zebra, modelos celulares, modelos 3D- recriar complexidade bioq]
Immunoelectron microscopy
Antibodies labelled with clusters of electron dense metal (gold) in electron microscopy
blocking solution–> Antigen –> primary antibody –> gold particle conjugated to secondary antibody
