Lecture 5 Flashcards
describe the difference between self and non self?
what is self tolerance
Self (mhc marker class 1)- tolerated by the immune system that distinguish you as self
Antigen (non self marker) - foreign protein is recognized as non self and is attacked
self tolerance (immune tolerance) - ability of immune system to remove foreign cells but not our own
what are Pathogen-associated molecular patterns (PAMPS)
how immune system recognizes non self antigens
-molecules shared by related microbes that are NOT FOUND on/in human cells
-immune & epithetical cells with pattern recognition receptors ON or INSIDE them are called TLR
-TLR can bind to many PAMPs and PRESENT them to the immune system or start a chemical reaction as a response to inflammation
examples : LPS, porins, peptidoglycan, lipoteichoic acids, flagella, bacterial or viral DNA or RNA & mycolic acid
what are DANGER –ASSOCIATED MOLECULAR PATTERNS
(DAMPs)
-found on stressed, injured, infected, or transformed human cells
-heat shock proteins and altered membrane phospholipids
-recognized by pattern recognition receptors
-initiate immune response and can be the CAUSE of autoimmune response
what are cytokines
-signal to molecules to initiate cell to cell communication.
* Mediate & regulate immune response & inflammation
* Stimulates Natural killer
(NK) cell production
what are chemokines
-kind of cytokine that stimulate the movement of immune cells towards sites of inflammation,
infection and trauma
what is an antigen
-formation of AB if when introduced the person doesnt have it
- from inside the body (self- cell) or outside (non-self)
-can be whole non-self cell like a bacteria or virus - Or a portion of a protein from an organism
-binds to AB - lock and key
-ABs not made toward the whole AG molecule just a specific part of it - EPITOPE
-AG ARE LARGE so B lymphs make ABs specific to many epitopes
POLLEN OR SPLINTER - Ag
what is a hapten
-Ag that can be bound by a matching Ab but is too small on its own to elicit an immune response
-must attach to a carrier molecule
-POISON IVY
what is an antibody
-Immunoglobulin
-glycoprotein formed by b lymphs in response to be being stimulated by an AG
-have specific binding sites on Variable - FAB portion that matches epitope (variable light and heavy chains)
-Fc portion -crystallizable - binds for phagocytosis
-12 domains with 2 heavy and 2 light chains
-light chains have cysteine with disulfide bridges
-constant amino acids (c) in light chain
-if the epitope is altered (mutation or genetic recomb) it will not fit the AG or bind with FAB
-viruses can evade AB defense by producing altering epitope that previous AB no longer fit
what is the immune system
- made up of organs, tissues, cells & molecules that protect against pathogens
-resist invasion, removed unwanted substances, neturalize toxins or SURVEY/PATROLL for foreign material
-made up of innate (natural fast acting non specific, no memory) and adaptive immunity (acquire through life and exposure, 2nd line, slower, specific, longer lasting immunity) WORKING TOGETHER - BOTH HAVE CELLS THAT CAN TELL BETWEEN SELF AND NON SELF
what is innate immunity made up of
1st line of defense (Barriers to entry)
* Skin -mechanical barrier
* Hairs in the nose, cilia of the respiratory tract
* Tears & saliva (lysozyme)
* Mucous membranes
* Acid pH of stomach and in urine
* Peristalsis
* Urine flow
2nd line of defense (Defense from invasion)
* Phagocytic cells: dendrites, eosinophils, basophils, neutrophils, macrophages & monocytes – engulf foreign material
* Natural killer lymphocytes-
* Cytokines/Chemokines
* Complement:(start off as inactive plasma proteins) increases # of phagocytes & phagocytosis, can lyse pathogens, opsonization- induce inflammation also found in adaptive
adaptive immune response - HUMORAL immunity - AB FORMATION
-mainly B cells (lymphocytes)
-*B cells have antibodies (Ab) on surface that bind to a specific extracellular antigen (Ag) and engulfs it
- fragmented Ag + MHC II are presented on B cell surface to helper T lymphocytes which bind using CD4 receptor
-Causes the release of interleukins which activate B cells to divide and become plasma B cells which release specific AB into the plasma OR become memory B cells so next time the ABs are made faster
adaptive immune response - Cell mediated response (eliminates infected or cancerous human cells)
-T cells (lymphocytes) – T helper cells and Cytotoxic T cells (& some B cells)
-Acts on human cells that have intracellular infections with
bacteria or virus or cancerous cells
-T helper cells have CD4+ receptors that bind to Ag presented on MHC II
-Cytotoxic T cells have CD8+ receptors that bind to Ag
presented on MHC I
-response can be initiated by antigen presenting phagocytes like macrophages, dendrites or B cells or DIRECTLY directly by a cell infected with a bacteria/virus or from a cancerous cell
-response ends with T regulatory cells
what are the 5 IMMUNOGLOBULIN CLASSES
-class of proteins present in serum, on immune
cells or in secretions which function as antibodies
-IgM, IgG, IgA, IgD, IgE
-distinguished by constant regions in their structure
-where they concentrated in the body
-half life
-function
-when they appear after exposure to foreign substance
IgG
major immunoglobulin in serum
-HALF LIFE 23 days
-diffuses readily into the extravascular spaces & neutralizes toxins, binds to microorganisms in extravascular spaces
-can cross the placenta
-IgG in cord blood and spinal fluid
IgM
largest in size which confines it to blood produced FIRST in immune response
-PENTAMER with J chain
-effective in agglutination and cytolytic reactions
-some IgM in cord blood; not detectable cerebrospinal fluid
IgA
predominant Ig in secretions: tears, saliva, colostrum, milk, intestinal fluids
-DIMER with secretory component with J chain
-synthesized by plasma cells on body surfaces and intestinal wall
-binds to secretory component in intestine to protect IgA from proteolytic digestive enzymes (secretory IgA)
IgD
-very low concentrations in plasma; less than 1%
extremely susceptible to proteolysis
-cell membrane Ig found on the surface of B lymphocytes in association with IgM
IgE
-trace plasma protein - THE LEAST IF YOU DONT HAVE PARASITES
- invading parasites/worms
-mediates hypersensitivity (allergic) reactions, allergies, anaphylaxis
-binds strongly mast cells and basophils; mediates release of histamines, and heparin
First exposure & response to a new antigen
-IgM made first and lasts about 2 weeks
-At around 1-2 weeks, IgG starts to appear (sometimes IgA as well). IgG increases for a month more than IgM
-Detection of high serum levels of IgM without IgG or with low levels IgG = recent first-time infection
SECONDARY IMMUNE RESPONSE
TO INFECTION
-AB production second or subsequent exposure to same antigen - AMNEASTIC RESPONSE
-rapid response of antigen sensitive memory B cells – Abs produced much quicker
-more IgG
-longer elevation gradual decline (AB catabolized)
IgG without IgM or very little IgM = past infection, second infection or vaccination
what is serology
Identification of Ab to an infectious agent or the specific Ag
-if the AB or AG is present it means infection
when do you do serology
-*diagnose an infectious diseases
* determine if an infection is recent or from the past
* confirm immunity to an infectious agent
* check blood type
* Diagnose patients with immune deficiencies
* Diagnose autoimmune disorders
how are AG-AB reactions in serology described
Specific reaction
Cross reacting
Affinity
Avidity
Direct Antibody Reaction
Indirect Antibody Reaction
Specific reaction
-Abs that only recognize and attach to the Ag that stimulated their production
Cross reacting
Ab that binds to an antigenic
determinant that is similar but not identical to the antigenic determinant that stimulated its production
Affinity
strength of the bond between the antigen’s epitope and the antibody’s paratope at a SINGULAR binding site (one arm of the Y)
Avidity
measure of the total binding
strength of an antibody at every binding site (affinity and number of binding sites)
Direct Antibody Reaction
Ag-Ab react directly to each other giving a visible reaction
Indirect Antibody Reaction
Addition of a 2nd Ab prepared against the species immunoglobulin (antihuman IgG) or using a latex bead to make the Ag-Ab reaction more visible
ab with fewer binding sites high affinity low avidity
Two important parameters in serological tests
Sensitivity (true positive):
Specificity (true negative):
Sensitivity (true positive):
- ability of the test to detect the smallest amounts of Ag or Ab
-how well a test can identify true pos results
-when a test is highly sensitive - false positives
Specificity (true negative):
-Ability of the test to detect a population of Ab molecules that react with only one Ag
-Ab only combines specifically with one epitope
-high specificity - false pos rare
-low specificity = lots of cross reactions (many epitopes) causing false positives
what are the different types of AB
Monoclonal Antibodies
Polyclonal Antibodies
Heterophile Antibody
Monoclonal Antibodies
-Ab’s made from a single B cell lineage from an immunized animal
-Single lineage B cells are removed from the spleen of animal and fused to an immortalized (myoloma) cell line
-produce Ab’s that are highly specific (from cloned b cells) and react only to a single epitope of the antigen
-no precipitation
Polyclonal Antibodies (right from animal blood and purified)
-* Heterogeneous Ab’s produced from multiple B cell lineages (many injections) from an immunized animal
* The Abs can react to several epitopes of the antigen
-lattices or aggregates occur
-precipitation occurs
Heterophile Antibody
-Abs that can react with proteins across species lines – can cause agglutination of red blood cell from other species as well
* Can cross-react with self-antigens and cause significant
interference
law of mass action
- Ag AND ab complexes are reversible
-rate of chemical reaction is directly proportional to the concentration of substances reacting
-equilibrium is when the number of bonds being broken are equal to those being formed
false serology why would negative serology happen on an infected person
Reasons for a positive serology result on a non-infected patient
a)immunocompromised -
* Serum collected too early in the infection – usually it takes 10- 14 days for Ab to show up
* Antibodies may not rise until months after acute infection -Lyme disease, Legionnaire’s
b)* Production of cross-reacting antibody or heterophile antibody
* Reactivation of latent organism due to infection by a different pathogen → Influenza A may cause reactivation of CMV
* Passive transfer of multiple antibodies -Patient receiving intravenous immunoglobulin
what are AB TITERS
what is Seroconversion
- Test that detects the presence of and the amount of Abs within a person’s serum
*Early infection more AG than AB
-Abs dont come in until 2-3 weeks after infection and can be delayed depending on the infection - Seroconversion = the time during which a specific Ab
develops and rises to detectable levels in blood
*in order to determine when you got the infection you test paired samples, acute and convalescent sera - One sample from ACUTE-phase (within 1 week of infection) and one sample from CONVALESCENT-phase (2-3 weeks later - recovery period)
-make doubling dilutions of pt serum -AB DILUTION and then add constant AG to each tube
-see how many times you can add the same AG and still dilute - Run test in parallel → acute serum and convalescent
serum
how to understand AB results
- Look for the highest dilution(MOST DILUTED) of a serum sample in both the acute and the convalescent set that causes a positive test reaction
- Lack of Ab in either set = no exposure or exposure is too recent
*having no Ab present in the acute sera
to Ab present in convalescent sera = the development of antibodies in response to immunization or infection - A four-fold rise in titer between acute and convalescent serum = an active infection
- If titer is the same in both tubes indicates the Abs were already in the serum and not recently produced = an old infection or vaccination
agglutination reactions TWO TYPES
sensitization
* Single Abs bind with antigen
* Happens by accident -Ab must “bump” into the cell at the corresponding antigen site
* forces that hold Ab and Ag together are weak
* Reaction is rapid and reversible & not visible- no agglutination
- Lattice formation
* Most Ags are multivalent- have several epitopes to where Abs can bind
* Abs have at least 2 antigenic binding sites -IgM ~10
*SLIDE PIC- the Fab portion of Ig molecule attaches to antigens on 2 adjacent cells (Y-Y-Y)
* Large lattices of Ag-Ab complexes are
formed
* Agglutination becomes visible result
* If Ag & Ab are soluble the visible reaction is called precipitation
types Agglutination reactions
Direct - Active
Direct active - hemagglutination
indirect - passive
indirect passive hemagglutination
reverse indirect -passive
DIRECT (Active)
* Antigen is the natural carrier particle
* Epitope on the organism reacts with the Ab and becomes visible as agglutination
Example: Serology for Salmonella
DIRECT (Active) Hemagglutination
* Antigen is intrinsic part of red blood cell
* RBC reacts directly with the Ab and becomes visible as agglutination
Example: ABO grouping
INDIRECT (Passive)
* Ag is artificially attached to a latex bead
* Detects Ab in sample
* Ag-Ab complex made visible by latex
Hemagglutination (INDIRECT Passive)
* Ag attached to red cells
* Detects Ab in sample
* Ag-Ab complex made visible by RBC
REVERSE INDIRECT (Passive)
* Ab affixed to latex or RBC
* If attached to a RBC then called Reverse Hemagglutination
* Detects Ag (like bacteria) in sample
SEROGROUPING OF ENTERIC PATHOGENS
- Salmonella, Shigella or Escherichia coli O157:H7 require serological testing to confirm the tube
biochemical identification - Serological tests for enteric pathogens are considered direct agglutination tests
- No latex beads or carrier molecules involved
cant use it right away because it can cause cross reactivity
COAGGLUTINATION
(a reverse indirect agglutination test)
Intact formalin-killed Staph aureus is the carrier
* Protein A in cell wall of Staphylococcus aureus, can bind to Fc region of most IgG antibodies
* Fab region of IgG free to interact with antigens present in the specimen
* Visible agglutination of S. aureus particles = antigen-antibody reaction
pathoDx for lancefield grouping
*Serological classification of Streptococci as per
the polysaccharide antigenic nature of cell wall
* BH strep can be serogroups A, B, C, F, and G
*NH strep is group D
* AG extracted form streptococci
- Reverse indirect agglutination test since blue
latex particles pre-coated with group-specific
antibodies react with antigen
staphurex
- Use of Latex particles coated with plasma containing IgG and fibrinogen to detect clumping factor
and Protein A on Staph aureus cells - Protein A reacts with the IgG clumping factor reacts with fibrinogen
- Since there are latex particles coated with antibody (IgG) & you are detecting Ag (the Staph) then
this is a reverse indirect agglutination test
coagulase
*if organism can make coagulase enzyme
that when mixed with rabbit plasma produces clumping or a clot. Two types of coagulase produced, bound & free
Bound coagulase
* clumping factor, found on cell wall of S. aureus
* Reacts with fibrinogen and causes agglutination type
clumping
* Detected by slide and tube coagulase tests
Free coagulase
* Extracellular coagulase excreted by cell
* Produces a thrombin-like substance that converts fibrinogen to fibrin (clot formed)
* Detected by tube coagulase only
hemagglutination inhibition HI***
- viruses like Rubella have surface Ags that can
agglutinate RBC
*reaction can be inhibited by Abs found in a person’s
immune or convalescent sera - Virus can be bound by the antibodies present in the serum therefore cannot agglutinate the RBC
Method:
* Serial dilutions of patient sera (Ab) are made
* A standard amount of virus (Ag) is added
* Add red cell suspension & incubate at room temp for 60min
* Highest dilution of serum that inhibits hemaglutination is the hemagglutination titre
a pellet is not a reaction the whole cell will be a hazy red
- Sera + virus +Ab (bound virus) + RBC = No agglutination or HI - LOOKS LIKE PELLET
*The highest dilution of serum that prevents hemagglutination is called the HI titer of the serum - Sera + virus - NO AB (free virus) + RBC = Agglutination
- Then hemagglutination will be observed in all wells =- HAZY ALL RED
precipitation reactions
- Soluble Ag and Ab combine to form lattice (a 3d structure formed by the reaction of polyvalent antigens with antibodies)
- WHEN concentration is optimum lattice falls out of
solution & forms a visible insoluble precipitate - Precipitate that remains suspended as floccules =
Flocculation
-can take place in liquid media or in gels such as agar, agarose or polyacrylamide - Precipitation curve= you can plot how much a
constant amount of Ab is precipitated when you add
increasing amounts of Ag
ON THE CURVE
Prozone:
* Ab excess
* Saturation of antigenic sites with antibody
* The Abs do not bridge between antigens, no lattice occurs
Zone of Equivalence:
* Ag and Ab in equivalent proportions
* Produces lattice formation
Postzone:
* Antigen excess allows a number of antigens to bind to a single antibody (excess antigen binding sites)
* Number of crosslinks decrease, prevents lattice formation- Can cause false negatives
OUCHTEROLONY QUALITATIVE PRECIPITATION
DOUBLE IMMUNODIFFUSION ***
- If trying to detect whether a patient has a certain Ab or Ag
-corresponding Ag in one well & patient sera in an adjacent well (or a known Ab in one well and patient specimen in the other)
* Both diffuse out towards each other in gel (double diffusion)
* If Ag-Ab reaction happens -get a line of precipitation at the optimum concentration
- If you want to test whether a patient has a specific Ag you can do a test of relatedness
- Place a known Ab to the Ag you are testing for in a center well
*a known Ag in another well (positive control) - Place patient specimen in another well adjacent to the positive control
- Compare patient result line to result line of know Ag for relatedness
- Called the pattern or line of identity
radial immunodiffusion
quantitative
* Single diffusion test since 1 of the reactants known Ab is evenly distributed throughout the gel
* Sample you are testing for is added to wells cut into the agar
* If Ag present it will diffuse into gel & form a precipitin band in a radial pattern, where equivalence occurs
* Diameter of ring is measured- amount of Ag is proportional to area of precipitation ring
* Standards of known Ag are also tested & a standard curve plotted
* Unknown sample diameters also plotted on graph
* Based on where they fall on graph can determine the
concentration of Ag in each sample = quantitative
AS THE SIZE OF RING INCREASES SO DOES THE ANTIGEN
WHAT IS A label immunoassay and what are the two types
uses of the binding between an Ag and its homologous Ab in order to identify and quantify specific Ag or Ab in a sample
Label:
* Chemically links Ab or AG with label to make the reaction detectable
* Labels emit radiation, produce a color change in a solution or fluoresce under light
- Two main types of Labeled Immunoassay are:
- Immunofluorescence
- Enzyme Immunoassay
DIRECT FLUORESCENT Ab (DFA)
- Uses one Ab to identify target Ag- can be on a slide
- Antigen-specific fluorescent-labeled Ab is
applied, incubated, washed and examined with
fluorescent microscope - Can detect infectious agent inside cells or cell cultures
NDIRECT FLUORESCENT Ab (IFA)
To detect target Ag in patient specimen
* Patient sample on slide
* First add unlabeled (primary) Ab that specifically binds the target Ag if it is present in the sample
* Then add the secondary Ab (usually an anti species Ab like anti-human Ab), which is labeled to the fluorophore
* The secondary Ab recognizes the primary Ab and binds to it
To detect Ab in patient serum
* Bacteria or virus fixed on slide – add patient sera
* If Ab present in sera will attach – add Anti human Ab
with fluorescent dye
DOUBLE INDIRECT IMMUNOFLUORESCENCE
- Uses a second, unlabeled antihuman Ab to bind to the microbial specific Ab in the patient serum
- Then a third fluorescent labeled Ab
directed to the second (antihuman Ab) is added
ENZYME LINKED IMMUNOSORBENT ASSAY
(ELISA)
- Uses enzymes (alkaline phosphatase or horseradish
peroxidase) conjugated to Ab or Ag - When substrate added if there is an Ag-Ab complex a
color will be produced that can be quantified
spectrophotometrically - wells in a microtitre tray or spherical plastic or
magnetic beads
*can be used to detect AB OR Ag in pt samples for direct or indirect tests
Direct ELISA for Ag DETECTION
* Patient specimen is absorbed to bottom of well
* An enzyme LABELED Ab specific to the target Ag is added
* If patient sample has Ag then will bind to Ab
* Add substrate and a color change indicates positive result
Indirect ELISA for Ag DETECTION
* Patient specimen is absorbed to bottom of well
* An UNLABELED Ab specific to the target Ag is added
* If patient sample has Ag then will bind to Ab
* Add second enzyme labeled species specific Ab (antimouse Ab) which will bind to the first Ab
* Add substrate and a color change indicates positive result
SANDWICH METHOD ELISA DIRECT AND INDIRECT ANTIGEN DETECTION
Direct Sandwich Antigen detection:
* Known Ab captured or bound to matrix
* Add patient sample – if Ag is present will bind to Ab
* Add secondary Ab to that Ag labeled with enzyme
* Substrate added and a color change indicates Ag-Ab reaction
Indirect Sandwich Antigen detection:
* Known Ab captured or bound to matrix
* Add patient sample – if Ag is present will bind to Ab
* Add secondary Ab to that Ag
* Add third Ab (anti human Ab) labeled with enzyme, that binds to primary Ab
* Substrate added and a color change indicates Ag – Ab
reaction
INDIRECT ELISA FOR AB DETECTION
Only Indirect ELISA for Ab detection:
* KNOWN Ag fixed to bottom of
well or tube
* Add patient sample – if Ab present will bind
* Add second Ab (anti-human) labeled with enzyme
* Add substrate and color change indicates positive
reaction
WESTERN BLOTTING
COMPLEMENT FIXATION
- Demonstrates presence of antibody in a patients serum
- Consists of mixing antigen suspected of causing patients disease + patients serum
- Limiting amount of complement is added
- If serum has antibody to antigen these complexes will bind all the complement
- Sheep RBC + hemolysin (indicator) added
- Only if complement has not been bound by Ab-An complex will it be available to bind to sheep cell hemolysin complex and cause lysis
Positive: failure of RBC to lyse in final test system
Negative: lysis of indicator cells = lack of Ab
TWO SYSTEMS
Test system 1 - nonimmune pt serum WITHOUT SPECIFIC AB, test AG + complement , complement NOT fixed
Test system 2 - IMMUNE pt serum with SPECIFIC AB, test AG + complement, complement FIXED
both go into the INDICATOR SYSTEM - sheep rbcs, guinea pic complement, rabbit hemolysin
TEST SYSTEM 1 -hemolysis negative CF AB test (pale)
TEST SYSTEM 2 - NO hemolysis - Pos CF antibody test
