CI 2 LEC 3 - Laboratory Evaluation of Immunoglobulin Function and Humoral Immunity

- y-shaped glycoprotein

immunoglobulin (Ig)

2 sites of an Ig

1. Fab region

2. Fc region

- antigen-binding site

- neutralization

Fragment antigen-binding (Fab) region

- site for complement activation/ cell surface receptors

- opsonization

Fragment crystallizable (Fc) region

holds the 2 polypeptide chains

disulfide bonds

- 110 amino acid residues at the N-terminal end

- 1 VL domain

- 1 CL domain

Variable region

- 1 CL domain

- 3 or 4 CH domain

constant region

5 Ig heavy chain isotypes?

2 Ig light chain isotypes?

IgM, IgD, IgG, IgA, IgE

kappa, lambda

Ig has 2 identical antigen-binding sites

bivalent

forms when there are >= 3 antigenic determinants

lattice

effects of lattice formation

- precipitation

- enhanced phagocytosis (opsonization)

- complement system activation

- B cell activation

provides flexibility due to hydrophilic amino acids and proline

hinge region

due to the hinge region, Ig is susceptible what enzymes?

papain

pepsin

Papain yields what?

2 Fab fragments

1 Fc fragment

Pepsin yields what?

1 F(ab')2 fragment

1 Fc fragment

stages of B-cell response?

1. antigen recognition

2. activation

3. proliferation

4. differentiation

Naive B cells (IgM⁺, IgD⁺) bind antigens via BCR

antigen recognition

Signals from antigen + Helper T cell activate B cell

activation

activated B cell clonally epands

proliferation

- secrete antibodies

- early response = IgM

- late response = IgG

plasma cells

- long-lived

- rapid response upon re-exposure

memory B cells

- affinity maturation

- high-affinity IgG

High-affinity Ig-Expressing B cells

4 weak noncovalent forces?

hydrophobic interaction

hydrogen bonds

ionic interactions

Van der Waals forces

"HHIV"

tightness of fit between an antigenic determinant and antibody binding site

affinity

aka. antigenic determinant (part of the antigen)

epitope

>/= 2 antigenic determinants

multivalent

2 antigenic determinants

bivalent

- single antigenic determinant & single binding site

affinity

- overall binding strength of the multiple binding sites

avidity

the increase in the average affinity of Ab produced as time passes after immunization

affinity maturation

remain bound after adding chaotropic agent

high-avidity antibodies

dissociate and are not detected after adding chaotropic agent

low-avidity antibodies

- differentiates recent infections vs. previous infections

- IgM (not reliable)

measurement of IgG antibody avidity

size of antigen-antibody complexes depends on what?

1. Antigen valence

2. Relative concentrations

cross-linking of multivalent antigen by bivalent antibodies

Ag-Ab precipitation

no cross-linking

monovalent Ag

linear chains or small cyclic complexes

bivalent Ag

forms large 3D lattices

multivalent Ag

Only precipitate antigens with repeating identical determinants

monoclonal Ab

- Recognize different epitopes on the same antigen

- cross-linking

polyclonal Ab

- Ab conc. = Ag conc.

- largest complexes

Zone of equivalence

Excess antibody

prozone

(if Henry's = postzone)

Excess antigen

postzone

(if Henry's = prozone)

 Humans can produce at least ___different antibody molecules

10⁶ to 10⁹

Constant, Variable, and Joining gene segments

Light chain

Constant, Variable, Diversity, and Joining gene segments

Heavy chain

heavy chain = Chromosome ___

kappa light chain = Chromosome ___

lambda light chain = Chromosome ___

heavy chain = Chromosome 14

kappa light chain = Chromosome 2

lambda light chain = Chromosome 22

- Confined to the variable regions of H and L chains, and the introns

- 1 mutation occurs per cell division 

- B-cells

somatic hypermutation

somatic hypermutation results?

- diversity

- changes in affinity

- selective

- high-affinity

- more effective Abs

 All B cells initially make IgM antibodies → IgG antibodies

class switching (idiotype)

Ensures each B cell expresses only one allele of light and heavy chains

Allelic exclusion

- primary antibody response

- First Ab made by B cells

- pentamer (aka macroglobulin)

IgM

present in autoimmune disorders (like SLE)

monomeric 7s IgM

IgM sedimentation rate = ___

IgM molecular weight = ___

IgM sedimentation rate = 19S

IgM molecular weight = 900 kDa

- 10 antigen-binding site

- highly efficient

- activates complement system

- cross-linking → lattice

- does not cross placenta

pentameric IgM

- rich in cysteine

- disulfide bonds: links Fc regions → pentamer

J (joining) chain

- most studied Ig

- major Ab class in blood

- secondary immune response

- activates complement system, but needs 2 molecules

- crosses placenta

- high diffusion coefficient

IgG

IgG number of molecules requires to activate the complement system

2

Significant quantities of IgG is transferred at ___ AOG

~12 weeks’

Adult IgG levels are reached at___ years old and remain constant

7

Fc region of IgG functions?

- opsonization

- antibody-dependent cell-mediated cytotoxicity (ADCC)

Dominant IgG subclass in adult humans

IgG1

IgG subclass that does not cross placenta

IgG2

Most effective IgG subclass binder of complement

IgG3

IgG subclass that does not bind complement

IgG4

most to least effectivity in binding complements

IgM > IgG3 > IgG1 > IgG2

IgG subclass: complement-fixing capacity via classical pathway

IgG1, IgG3

IgG subclass: negative for heterologous skin-binding capcity

IgG2

IgG subclass: negative for macrophage receptor

IgG2

IgG subclass: negative for reaction with protein A

IgG3

IgG subclass: positive for Anti-factor VIII

IgG4

IgG subclass: positive for:

- antidextran

- antilevan

- antiteichoic acid

IgG2

IgG subclass: highest number of inter-heavy chain disulfide bonds in hinge region

IgG3 (5)

major Ab in secretions

IgA

IgA 2 forms?

- monomers (4 chains)

- dimers (2 monomers)

IG classes containing J chains

- IgM (pentamer)

- IgA (dimers)

"JAM"

IgA secretory component (SC)?

70 kDa glycopolypeptide

cells that produce secretory IgA

- plasma cells

- epithelial cells

protects IgA from proteolytic enzymes 

secretory component

dominant IgA form in secretions, though a minor component in serum.

IgA2

intestinal IgA system may be fully developed

2 years old

Serum IgA reaches adult concentrations

12 years old

- 1st line of defenses → external environment

- inhibits adherence

- reduces allergic reactions

- alternative pathway

- opsonin

- induces eosinophil degranulation → antiparasitic response

IgA functions

- minor component in serum

- major membrane-bound Ig on the surface of B lymphocytes (newborns)

IgD

- turning on/off/modulating (controls) B-cell division or differentiation (immunoregulation)

- No specific biological role as a humoral Ab

IgD

- detected at 6 months; remain low throughout life

- increased in chronic infection

- no specific disease association

- normal in allergies/ autoimmune disease

- absent in hypogammaglobulinemia

IgD

- lowest serum concentration

- homocytotropic antibody (attach to human skin)

- reaginic antibody (initiate allergic reaction)

- bifunctional

- produced in the respiratory & intestinal tract linings

IgE

contains the Fc region,

H (hinge) region

___: Found in blood and tissue fluids

___: Mainly in the blood

___: On epithelial surfaces

___: Bound to B cells

___: Bound to basophils and mast cell

IgG: Found in blood and tissue fluids

IgM: Mainly in the blood

Secretory IgA: On epithelial surfaces

IgD: Bound to B cells

IgE: Bound to basophils and mast cell

sex with lower IgM

males

have higher Ig levels

Darker-pigmented Individuals

-  clonal expansion of a single Ig-producing lymphoid cell

- for a particular antigen

- restricted heterogeneity

monoclonal Ig

screening test for monoclonal gammopathy

protein electrophoresis (serum or urine)

monoclonal gammopathies?

- multiple myeloma

- Waldenstorm macroglobulinemia

- Burkitt lymphoma

- B-cell neoplasms

- B-cell ALL

Clinical syndrome due to interaction of an IgM paraprotein:

- Myelin-associated glycoprotein (MAG)

- Gangliosides

Paraproteinemic neuropathies

 motor neuropathy?

sensory neuropathy?

GM1

GMd1b

- 75% of organ transplant patients

-  immunosuppressive drugs

Transient monoclonal gammopathy

Monoclonal gammopathy present but (–) no malignancy features

Monoclonal Gammopathy of Undetermined Significance

differentiate with multiple myeloma

urine Bence Jones Protein

- Structurally different Ig

- expansion of multiple Ig-producing lymphoid cells

polyclonal immunoglobulins