Complement Cascade * Classical Pathway = response to antibodies * Alternate Pathway = response to microbial infection 1. Membrane attack complexes = lyse membranes 2. Chemoattractants = attract phagocytic cells 3. Opsonizes or coats bacteria = enhancing phagocytosis

• Note that complement is inactive in bloodstream; they become activated enzymes when needed during an immune response

soluble proteins found in the blood vessels...aids in destroying the invading protein. * opsonization-complement protein coats the outer surface which allows microphage to recognize and engulf much easier. * Membrane attack complex- complement proteins makes a hole in pathogen resulting in lysis. Complement proteins circulate in an inactive state in blood stream until they encounter a pathogen.

• has no specificity
• inactivates @ 56 degrees c  / 30 minutes

player in immunity Major Histocompatability Complex (MHC)

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• phagocytosis -monocytes are found in bloodstream
•  macrophages are found in tissues 
• these are antigen presenting cells 

Macrophage Funtions also include secreting molecules such as IL-1 and TNF

Macrophages cell markers: HLA class II, IgG receptor, and C3 receptor 

genetic variations in the Ig struture; located in the constant region; only varies by a couple of A.A. My IgM may be different from your IgM. 

Variation in the constant region of the Ig

site on antigen that identifies it as foreign. As in blood A. A= antigens on RBC

H=O blood which would be antigenic  cancel that thought O is universal donor

So A blood transferred to and O blood recipient would be immugenic to the recipient i think 

Interleukin-1 (AKA cytokine) mediates activation of __________.

Interleukin-2 (IL-2) enhances activation of more t cells. The latter is

producted by the t-cells. 

What prompts this response? 

T-4 helper cells.

Thymus dependent response ('double hand shake') involves Protein antigens

 Im guessing because these are more complex and stimulate greater immunogenicity. 

Thymus Independent Humoral Antibody Response

What type of antigen is involved?

• long repeating polymers as in Carbohydrates and glycoproteins

• B-cell activation directly by antigen(no T helper cell needed)

• Ab response primarily IgM

• No MEMORY; No future protection

What mediates the activation of T4 cells during

the thymus dependent response?

Interleukin-1

(Produced by monocytes and macrophages)

Which T cells (4 or 8) control and turn off the

antibody response?

The location on an immunoglobin that is

specific to the antigen:

Variable region

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serum * doesnt include fibrinogen

Side note: Recall in the lab, the serum (Ab) was clotted to prevent chealation of Ca+2...which complement is highly dependent upon.

Quantitative proceures to detect and measure immune response

Ehrlich

Thymus dependent response

Involves foreign _____

Depends upon ________

Keep in mind that this process is humoral immunity..the Ab-dependent

Proteins; t-cells

1. Macrophage engulfs and presents MHC II Ag
2. T-4 aka cd4 recognizes MHC II Ag
3. IL-1 activates t-4 cell
4. IL-2 (produced by the tcells) promote division and increases the # of t-4 cells 
5. t-4 releases interleukins 2,4, 5,6, and 13 to activate b cells; activated bcells are plasma cells
6. plasma cells produce Ab's and memory cells
7. Ab and complement combine with Ag and destroy it
8. Macrophage clean up
9. T-supressor lymph (T-8 or CD8) control and turn off Ab response; stop Ab production process; prevents Ab production from over-utilizing

• (HLA-DP, -DR, -DQ)
• These are associated with humoral immunity
• These are found on macrophages (and other phagocytes) and present the digested Ag fragment on the surface of the cell.  
• Only T4 recognize this class provided the t4 receptor is specific for the APC

Which cells are able to proliferate sooner is response to an Ab, Memory, Naive T cells, or Naive Bcells

naive basically means these cells have not been exposed to Ag. Keep in mind the resting life for bcells is short (look up the life). The resting state for Tcells is several years. 

• direct killing of a foreign antigen by the cells themselves, without the aid of Ab production
• T-cytotoxic (T-8) and NK involved
• does not involve Ab; not phagocytosis,has specificity

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t4 secretes lymphokines:

-Macrophage chemotactic factor-Helps summon more macrophages to area

-Gamma Interferon

-IL-2 (this triggers growth of the T cytotoxic lymphs

-T-8 cytotoxic lymphs recognize the foreign antigen combined with Class I MHC antigen

-lysis of Antigen

Again CMI is the direct killing of a foreign antigen by the _________ themselves, and does NOT involve ______ production.

It involves cells that have ___________ pathogens or "issues" as opposed to humoral immunity. 

Examples: 

Virally infected cells 

fungi and bacteria

tumor cells

Parasites

Cells

antibody production

intracellular

1. 'summons' more macrophages to the area
2. involved in Cell mediated immunity
3. Secreted by the T-4 cells 

• not MHC class restricted;NK cells are not class restricted. If they don’t see MHC, they will attack!

• Antibody Dependent Cell-Mediated Cytotoxicity (ADCC) – direct killing of a target cell by way of an Fc receptor

• They express CD16

NK...Not are neither bcells or tcells

Express CD_____

If they do not see _____, they will attack

ADCC

Target cells coated with ______ by way of ____ receptor for ab on the NK cells  

If  IfdonIf they don’t see MHC, they will attack’t see  they don’t see MHC, they will attackMHC, they will attack

CD16

MHC

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**intracellular antigens (viral or tumor)

MHC II

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Subclasses

Ig has ___

IgA has _____

IgM_______

All of the immunoglobins have 2 binding sites

(monomers) except ____ which has ____

IgM which has 10 but can only use 5 due to

steric hinderance (is a pentamer)

Although this immunoglobin cannot pass

through the placenta, it is found in the mother’s

milk. Which immunoglobin am I?

• The DNA is rearranged during blast formation to make very Specific antibody

• Susuma Tonegawa – won the Nobel Prize in 1987 for describing this concept

Hinge Region & Bonds

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1. Hinge region: disulfide bond binds two heavy chains together

• The number of bonds varies which provides the molecule with flexibility

1. The light and heavy chains are held together also by disulfide bonds

IgG characteristics

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• 80% of total Ig (composed of mostly polypeptide and ~10% carbohydrate)
• Longest half life of 25 days
• 4 subclases (which vary according  to the # and position of disulfide bonds
• Macrophages, monocytes, and neutrophils have receptors on their surfaces that are specific for the Fc region of IgG
• Antibody dependent cytotoxic cell (AACC) Nk cell attaches to fc fragment
• Opsonization-coats cells bearing antigens to permit phagocytic cells to attach to Fc fragment

# of sites that Ag can bind to

• All isotpyes have 2 excpect for IgM

% Total Ig 

*increase to decrease % in circulation (lymph and blood stream)

Gansta MADE

IgG subclasses  

IgG III ___, ____ and ___best at complement activation 
(in order) 

IgG I and III Best at _________

•  IgG3, IgG1, & IgG2-complement activation in order of "best"
• IgG I and III-best at crossing the placenta

IgM 

Valence_____

Complement activation__________

Functions: 

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Valence= due to steric hindrance there's 5 unless ag's are tiny (then up to 10)

Complement: since it takes two basic ig units to activate complement, IgM does it very well because there are always 2 basic units in close proximity

Subclasses: 2

Functions: 

• 1st one produced in immune respose 
• 1st one a fetus is able to produce
• neutralization of toxins
• Complement/fixation and opsonization (4+)

• Immune adherance helps stick to walls
• Opsinogen-enables or heightens the binding process

Anaphylatoxin- 

which complements are involved?

• A substance that causes the degranulation of basophils thus releasing histamine and dilating capillaries
• c5a, C3a, C4a

• Anaphylatoxin and Chemotaxis

 __________ – discovered the alternative pathway of complement originally called – properdin system

Ions neccessary for complement to function

Ca2+ plays a role in?

Mg2+ plays a role in?

Ca2+ plays a role in the classical pathway in the “Recognition Unit”

Mg+2 also plays a role in cleaving c2... kinin activity of c2b

Mg2+ plays a role in the alternative pathway for complement activation

Complement activation 

IgG must be within __to ___nm of each other to be able to activate complement

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Classical Pathway recognition unit (see pic) C1q,c1r,c1s

IgG Molecules close to each other (30-40nm). Can also be just  ___ IgM

C1Q binds to ___ of Ig

C1r ONLY acts on C1s

C1r & C1s actualy bind together and calcium stabilizes this bond. With out calcium, the recognition unit would just fall apart.

FC; can be just one IgM molecule

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Complement Activation Unit resulting in _____ (see pic).

The cleaving of C2 results in C2a and c2b.

C2a binds with C4b with the help of ____.  C2b acts like ____ to increase vascular permeability

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C4b2a =convertase 3

Mg2+

kinin

C3 convertase cleaves to c5 convertase

*explain then refer to pic 

What is the activation unit_____

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Here is the C3 convertase on the cell

Remember, C3 is the most abundant complement protein in serum. It is even secreted by macrophages

C3 is cleaved into C3a and C3b.  {{{Animation Pivotal Step in the Process!!!}}}}

C3a becomes an anaphylatoxin

C3b binds to the the C4b2a complex. 

With the addition of ____, the activation unit is complete. 

Why is this bound more than once to the membrane?

What else does this molecule participate in? 

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C3b most abundant complement; cell adherance and opsinization

ADCC

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NK cell has Fc receptor that binds to Fc fragment of Ig. 

Macrophages and neutrophils can also destroy using same mechanism....target cells coated with ab...attach to fc receptor of leukocyte

Alternative Activation part of innate immunity.

What are some things that can activate it?

Affinity

ie...hydrophobic bonds, ionic bonds, van der waals....

Avidity

Cross Reactivity

 The sum of the forces binding the multivalent ag's to bivalent (igM of course has 5) ab's.

side note: The better the fit the stronger the attractive force= affinity.  

A structurally similar ag is capable of binding to an epitope, the affinity however will not be as strong. 

multiple

multivalent ag and bivalent ab

the aggregate # of multivalent sites of ag and ab are ~ equal

The concentrations of ag and ab that yield maximum binding = zone of equivalence

individual develops his/her own ab's by actual exposure to the ag is_____

Active immunity

the flu

active natural

made by someone else and given to a nonimmune individual

the ab's are short lived and there is no memory associated

(anti hep B)

Enhancing factors to bridge the gap include:

*bacterial cells and rbc's have slightly negative charge repel one another

*IgM has a diameter of 35mn...they are strong agglutins

-temp/incubation time

enzymes such as papain to neutralize charge and inhibit repulsion

centrifugation

pH 6.7-7.2

na+ concentration-again controlling the surface charge 

Chemicals such as polyethlene glycol

antigens found naturally (rbc's and bacteria cells?) on a particle. 

Include Hemagglutination 

Indirect Agglutination

 _____ bound to an inert particle

Detects _________

Exm: 

Uses:

latex beads serve as _______. 

 Antigen

Antibody

 ie...latex bead which would have them added onto the particle by a manufacturer

Group A strep Rheumatoid factor

carrier molecule and does not interact with the reaction

Zeta potential is a natural repulsive  ____ that occurs between ____ and even bacteria. This repulsion is due to: 

What is the distant between RBC's?

How does the saline contribute to the force?

What is the diameter of IgM?

What is the diameter of IgG?

force; RBC's

slightly negative charge on the cells which positions rbc's about 25nm apart; (the .9% saline (diluent) minimizes the repulsion by creating Na+ ionic cloud surrounding rbc's)

IgM is 35nm in diameter;therefore, this ab has the ability to brigde the gap without employing enhancement techniques.

IgG is 14nm-not large enough to enable binding

Agglutination assays are usually very  _______.

Antigen are typically fairly large particles and it takes  _______ antibodies to create a visible reactions

Reverse passive agglutination is used to detect an _______.

 _________ is bound to an _____ particle. 

These assays have great use for organisms that are either extremely difficult or dangerous to  _____

 Uses: _______

They can also be used to detect plasma proteins such as  ______or CRP – these are proteins and will have ____________

used to detect an antigen in a patient sample

 Antibody inert (something that doesnt interfere with rxn)

culture

Group a and B staph haptoglobin, c reactive protein

antigen sites too

Agglutination inhibition

Basically, if you add patient sample and antibody together, the two will combine following incubation IF the patient sample contains ______. If _____ are not present, the antibodies will have nothing to bind to.  

Next, antigen coated latex particles are added. The sample with bound ab and ag (drug present) will not bind to latex antigen; therefore no ________.    The sample with unbound antibodies will ____ to the antigen coated latex particles and result in _______. This is useful in detecting ellicit drug abuse.  

antigens; antigen

no agglutinatino = positive

bind;  with agglutination = negative

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Indirect Antiglobulin Test aka anti COOMB's test

isolate patients _____ then add reagent ______ = _______.

Add Antiglobulin test to enhance agglutination. 

this test primarily used to detect alloantibody testing for ______.

used to detect either ____or _____ on rbc

antibodies add reagent RBC=antibody coated cells

blood transfusions

ag's or ab's

Direct 

Anti-Human Globulin

ab coated RBC (patient sample) add anti globulin=agglutination

Coagglutination

protein A found in species such as S. aureus serve as a receptor for the fc portion of IgG. It holds onto this particular antibody which can then be used to detect the presence of an _____

This assay is highly specific using ______ carrier for human _____to bind to. 

antigen

bacteria ; antibody

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Anti-Human Globulin

Reagent  ______ that is designed to bind with the____Fragment of Human IgG

ab

fc

f pos

Heat inactivation of serum

Repeated freeze/thawing of samples

Auto-reactive antibodies

Passive Ig Administration

Maternal Ig

Severe hepatic disease

Some vaccines & malignancies

Technical error

False positives for Western Blot:

Infection with HTLV-1

Bilirubinemia

HLA antibodies

Polyclonal gammopathies

Autoimmune disease

Antigen Testing: p24

p24 antigen in the core of HIV-1

Detection & level is assumed to correlate with viral load

High during the initial infection (acute phase)

Undetectable during the latent phase

Return of p24 during AIDS Crisis

p24 disappears ~a week before we can detect p24 antibody

Formula: Abs # CD4 T Cells = WBC Count  x  %Lymphs  x  %CD4 T Cells

CD4 Count: 231

CD 8 Count: 501