Immunology and Plasmids Flashcards
What is the definition of an antigen?
a foreign material typically a protein that elicits an immune response so we can have specific recognition
What is the first line of defense?
Innate; skin to prevent entry, mucous membranes and their secretions (e.g. lysozymes) to prevent colonization; normal microflora to act as competition for nutrients; all non-specific
What is the second line of defense?
Natural killer cells and phagocytes; inflammation; fever (creates non-optimal environment, also helps immune cells to divide faster); antimicrobial substances; still non-specific
What is the third line of defense?
Adaptive (acquired immunity); specialized lymphocytes (T cells and B cells); antibodies
Describe the general characteristics of antibodies
Y-shaped molecule where each “prong” bind single, identical epitopes; composed of a light chain and heavy chain (V and C components, where C is elongated in the heavy chain but the V components remain the same size)
What are the different kinds of antibodies?
IgM (antibody, secreted first during immune response; snowflake shaped), IgG (main antibody involved in the immune resposne; single Y-shape), IgA (secreted antibody, two Y’s attached at the Fc), IgD (“dispensible”–does not seem to be essential), and IgE (involved in the allergic reaction…“evil”)
What are the characteristics of IgG?
Monomer, therefore bivalent, most abundant Ab (~80%), recruits immune system, can also be secreted like IgA, is present in the lymph, blood, and intestine, can cross the placental barrier, enhances phagocytosis; neutralizes toxins and virsuses; protects the fetus and newborn; half life of 23 days;
Random deletions of V, D, and J genes in the light and heavy chains create unique antibodies–no change in the C chain?; subsequent mutations of the already-created antibodies refine the existing ones
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What is the B-cell clonal selection theory?
Test 1: if the cell makes self-antigen binding antibodies, kill it
Test 2: if the cell makes antibodies which are useful in fighting the infection, stimulate it to divide to proliferate the antibodies
What are the forms of B cells and where do they come form?
Plasma B cells (protein factory–pump out the antibodies into circulation) and memory B cells (divide slowly and do not make as many antibodies; useful for long-term immunity); they come from naive B cells that are activated to germinate upon antigen exposure
Remember that when you are first infected with an antigen, your body will produce IgM Ab in small amounts peaking at 10-11 days, but it will produce IgG antibodies in much larger amounts peaking at about 15 days; as infection is taken care of Ab titer decreases; when reinfected, body still makes new IgM Ab but memory cells are converted to plasma cells and a much larger IgG Ab titer is created (peaks within 7 days of the second infection, as does IgM)
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What are the protective mechanisms of Ab joining to An in vivo?
Agglutination (antibodies bind the same antigen and clumps them together, reducing the number of infectious units to be dealt with); opsonization (coating An with Ab to enhance phagocytosis); neutralization (Ab blocks adhesion of virus and bacteria to mucosa and blocks the attachment/activity of toxins) ; activation of complement (builds pores into cell wall to causse cytoplasm to leak out); Ab-dependent cell-mediated cytotoxicity (used for fighting bigger antigens such as eukaryotes; Ab bind to target organisms and cause eosinohpils to release lytic enzymes to digest the organism)
What are secondary Ab?
Secondary antibodies bind to the primary antibody to assist in detection, sorting and purification of target antigens.
All mouse Ab specific for a certain Ag will have the same tail (crystallizable fragment, Fc) but will have different Ag binding sites due to random mutation
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How are Ab made in situ?
Lab animal (horse, hamster, mouse, rat, sheep, goat, chicken) are injected with a signle Ag (usually a protein, but may be a polysaccharide, hapten, etc) and will create Abs against it; serum is collected and Ab is purified; secondary Abs can be made from the Fc (secondary Abs specific for the Fc tail)
How are Abs collected from blood?
Blood from a lab animal is spun to precipitate cells down to the bottom; yellowish serum (plasma) is passed through affinity chromatography which binds the Fc region of Abs; elution occurs by a low pH (glycine, pH=2.4) and then neutralized in fractionation tube; samples are tested by SDS-Page to determine peak fraction
What are polyclonal Abs?
Polyclonal: all IgG are from different B cells
• Each antibody from a different B cell may bind a different epitope
○ Good, because if one epitope mutates, there may be several other sites that different antibodies can still bind to;
Immune reactions are polyclonal, which is beneficial because if one epitope mutates so the Abs from a B-cell no longer recognize it, the other B-cell Abs will still recognize their epitope and can fight the infection
What are monoclonal Abs?
Monoclonal: all from one B-cell, only bind one epitope
• Start off with a B cell and makes it so that it never dies
Abs made from different B-cells may bind to the same antigen, but on a different site?
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How are monoclonal cells produced?
B-Cells, of which only a few make antibodies, are fused with immortal cancer cells through Polyehtylene glycol and sendai virus, which will fused two eukaryote cells together if they do not have a cell wall; then, only the resulting hybridomas will survive selective growth medium; then they will be further screened and clone to ascertain the hybridomas that react with antigens; every Ab will be the same because it came from the same B cell?–test for the specific antibody?
What are the types of Ab labelling?
Isotope labelling (Iodine-125 or Sulfer-35 are attached to the protein; as they isotope decays, the electrons expose an X-ray film and the dark spots correspond to the presence of protein; enzyme labelling (common enzymes like HRP or alkaline phosphatase catalyze reactions that give a coloured product or chemluminesence); fluorescence labelling (a dye is attached to the protein that absorbs one colour and gives off another); may need to be visualized under UV light or may be visible on its own; heavy metals, which are good for electron microscopy
Why does fluorescence detection need a filter?
to block out any emitted light below a certain wavelength to limit interference
What are some western blot applications?
Detection and quanitification of potein in sample; if it was run on SDS-page, it can also give us information about the size; expression profiles of proteins in tissues (expression of diseased state vs normal state, eg); vrification of transgenic organisms; detect infections–see if antigens are present in the sample, OR anti-antigen antibodies are present
What is the transgenic expession example we studied in class?
RPE65 is a gene involved in recycling Vit A, which converted retinal from it’s inactive to active form to help see moving pictures; those who lack this gene are blind (?); package the gene into a virus and insert it into the eye
Describe an HIV test
ELISA western blot; nitrocellulose/nylon (?) sheet is covered with HIV proteins; the primary Ab are your own and will bind to HIV proteins; secondary Ab are goat-anti-human HRP (whcih will give off a coloured product); if two bands are present, you go to a moe specific test, because ELISAtest can show false positives
ELISA tests are similar to Western blots but do not cotain the protein separation aspect (SDS
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Describe the steps for an general ELISA test
Samples are placed into wells (microtiters) or blotted onto membranes; blocking proteins are added and washed away; primary (detection) Abs are added and washed away; secondary Abs linked to enzymes added, washed away; chromogenic reagents added, washed away
How does a pregnancy test work?
Pee on the stick; the urine will then move the mobile enzyme-linked MONOCLONAL mouse anti-HCG Ab; if HCG is present, it will bind to an IMmobile, POLYclonal, mouse anti-HCG Ab; mobile Ab will then bind to HCG on the immobile Ab and the enzyme will catalyze the colour reagent to a coloured product; any unbound mobile enzymes will stick to the immobile polyclonal anti-mouse Ab
Agglutination assay general description
Ab clump with Ag (precipitin reactio)–>confirms that both Ab and Ag are present; can test for the presence of: uknown cells using known Abs; OR known cells in an unknown serum (unknown Abs)
What are the possible identities in an ouchterlony test?
Non-identity–both wells contain different Ag that still ppt with the Ab; results in two lines that cross
Partial identity–both well contain the same antigen, but one well contains a second Ag which results in a spur pointing away from the sample containing the extra Ag
identity–shared, singular Ag in both samples which causes the ppt line to curve in a parabolic shape
How is an immunofluorescent stain carried out?
Cultured cells or tissues are fixed onto slides by paraformaldehyde/methanol/acetone, which will kill the cells; rinse; cells are permeabilizaed by treating with dilute detergents (which will open the cell membrane); cells are blocked to reduce non-specific binding (proteins stick to the blocking reagents and are rinsed away); primary Ab is added and rinsed away; secondary fluorescent Ab is added and rinsed;
What are the types of fluorescence microscopy and what are the pros and conns?
Standard FM: whole image is illuminated, out-of-focus light makes in-focus image fuzzy; Pros: cheap; Cons: fuzziness and photodamage
Confocal FM: whole image is illuminated and out-of-focus light is blocked; Pros: very good resolution; Cons: photodamage
Two-photon FM: Less energetic light is used (IR light) and two photons must hit the dye simultaneously to excite it; Pros: lower photodamage, better tissue penetration; Cons: costliest, less resolution than CFM
Regular agglutination requires cells to exhibit visible clumping, but ouchtrlony can showing clumping with proteins
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why do we not want to use polyclonal mobile Ab in the ELISA pregnancy tests?
Polyclonal would saturate all epitopes
• We could not get it to stop at the first trap position
• More HCG would be need to see a positive result
• Result in a loss of sensitivity
What is a conjugative plasmid?
Transmitted during conjugation; carry a variety of info
What is a resistance plasmid?
Protects against environmental factors; MDR (multiple drug resistance) plasmids
What is a colicinogenic plasmid?
Codes for proteins that kills other microbes (allows for availability of more nutrients)
What are degradative plasmids?
Contain genes for novel catabolic enzymes?
What is a virulence plasmid?
Increases the pathogenicity of an organisms (e.g. codes for toxins)
What is an F plasmid?
F+ or F-
• Allows cell to distribute daughter nuclei properply
• Also allows to make the sex pilus which allows for transfer to another bacteria (makes an F- cell itno a F+ cell)
• Whole genome is not being transferred
• Not in all bacterial species
• Selfish plasmid
○ Doesn’t really do anything for the cell,it just wants to transfer itself
Bacteriaa may contains hundreds of copies of a single plasmid to allow for rapid duplication and expression at high levels of genes conferring an advantage to them
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What are the typical components of a plasmid?
Independent ORI; capable of accepting the inserted gene; positive selection gene (only cells with the vector survive); Insert differentiation gene (cells with the insert look different than cells without); cloning sites (places to insert the DNA)
What is the procedure for western blot?
A polyacrylamide gel is first run (SDS, native, or 2D); the bands from the gel are transferred to a sheet of nylon or nitrocellulose (which have high affinities for protein); can transfer through two ways: capillary transfer, which is running liquid over the sheets, or electrophoretic transfer which is pulling proteins onto sheet through electrical current; since the sheets have high affinity for protein, blocking proteins (such as BSA or skim milk powder) must be added so the Abs only bind to the proteins of interest; the primary Ab is added and then the secondary Ab is added to detect the presence of the primary Ab; no specified type of Ab labelling
What is the reaction for HRP to make chemiluminsence?
HRP+hydrogen peroxide+luminol=unstable intermediate which gives off light (3-aminophthalate +N2 +light–425-510nm)
What kind of antibodies are involved in ouchterlony tests?
polyclonal antibodies
