module 03 section 01 (Igs) Flashcards
lymphocytes develop from a common stem cell through which process?
lymphopoiesis - the differentiation of lymphoid cells from a hematopoietic stem cell
what factors (generally) detmermine the multi-step process of lymphopoiesis?
singals conveyed by cell-cell interactions and soluble factors
what are the effector molecules of humoral immunity
immunoglobulins (antibodies)
immunoglobulins are glycoproteins, true or false?
if false what type of protein are they?
true
what are the two ways antibodies function (hint: where are the two places they can be found)?
they can be secreted by plasma cells (differentiated B-cells) OR serve as the antigen-binding receptors on b-cell membranes
what are the two types of antibodies?
monoclonal and polyclonal
describe monoclonal antibodies
antibodies that are derived from a single B-cell clone and are therefore specific for a single epitope on an antigen
describe polyclonal antibodies
a heterogenous mixture of antibodies with different anfinity produced by many clones of B-cells, recognizing multiple epitopes on antigens
what is the hallmark of our immune system?
the precision - one antibody specific for one particular microbe
what is the primary function of immunoglobulins?
to bind specifically to antigens (necessary for protection of the host)
how do antibodies act when expressed on the surface of B-cells?
act as receptors mediating antigen-triggered activation of the cell
how do antibodies act when secreted into the blood by plasma cells?
act as mediators of specific humoral immunity by binding to antigens
what are effector functions?
secondary function of immunoglobulins:
on top of their primary function, soluble immunoglobulins also elict a variety of biological responses through interactions with varius other cells (including phagocytic cells, mast cells, basophils)
list 2 effector functions of soluble immunoglobulins
(1) binding to t-cells as part of antibody dependent cell cytotoxicity
(2) binding to C1 complex in complement fixation, resulting in lysis of cells, release of biologically active molecules, and clearance of immune complexes in the blood
can the presence of a certain type of antibody be used as a diagnostic tool? why or why not?
yes - antibodies are produced in response to an infection, illness or irregularities in self cells
what is an ELISA test?
enzyme linked immunosorbent assay; a labratory test that detects the presence of a particular antibody in the blood of an individual through analysis of the antiserum
what is antiserum?
serum containing antibodies that bind to a particular antigen
when preforming an ELISA it is necessary to centrifuge the blood samples, why?
to precipitate the blood cells and obtain the clear fluid known as serum - any cells that remain will interfere with the assay and may influence results
what was the prodecure for the ELISA lab example in the module? (9 steps)
- blood samples were centrifuged
- blood samples were diluted (1:2, 1:10, 1:100) using buffer
-blood samples were added to the plate - positive control (anti-dna primary antibody) and negative control (buffer) are added to the plate
** everything was added in triplicate ** - incubate the ELISA plate at 37 degrees for 15 mins, then remove
(insures reaction occurs properly - 37 = body temp) - remove fluid from each well of the ELISA and wash with PBS
- add buffered solution containing secondary antibody that recognizes the antibodies made in humans
(secondary antibody is made in rabbits and has HRP enzymes) - incubate the plate again at 37 degrees for 15 mins
- remove fluid from each well and wash with PBS
- add buffered solution containing the chemical (HRP) substrate
- if there are human antibodies present the clear substrate will turn yellow
- time for 15 mins
- if all three triplicates are yellow - probably has it
- if only two - probably has it but requires more testing
- if none - probably does not have it
what is the ELISA result of a patient who’s results show that the antibody is present, however, the patient is not sick (as they may have already had the disease but recovered)?
true positive
what is the ELISA result of a patient that may be a poor producer of the antibody, or have some interfering substance in their blood, or the amount of anitbody is too low to measure accurately or goes undetected?
false negative
what is the possible ELISA result for a patient who has an unrelated antibody that reacts with the desired antigen non-specifically?
false positive
why are diltuions of sera used in ELISA testing?
results are based on color saturation of the enzyme attached to the second antibody, so using multiple dilutions helps gauge the level of antibody in the sample
why are ELISA tests done at 37°C?
what would happen if the test was done above 37°C?
what would happen if the test was done below 37°C?
because in order to obtain the correct results, the testing environment must mimic the environment of the human body, and body temp is 37°C
- too high: denaturation
- too low: reaction wont proceed
- either way would get a false neg
what is the purpose of the second antibody in the ELISA test?
detection of the antibody of interest (binds to the antigen-antibody complexes bound in the wells)
describe key features of the basic immunoglobulin structure
10
- 2 identical heavy chains (inner)
- 2 identical light chains (outer)
- interchain disulfide bonds
- variable region
- Fab region
- Fc region
- constant region
- hinge region
- domains
- oligosaccarides
what is the fucntion of interchain disulfide bonds in the immunoglobulin structure?
- hold together the light and heavy chains
- also exist btwn each of the polypeptide chains
describe the variable region of immunoglobulins
- variable domain, light chain (VL) = 110 aa
- variable domain, heavy chain (VH) = 110 aa
- top half of the Y arms
what is the Fab region?
- arms of the Y
- antigen binding fragment
what is the Fc region?
- base of the Y
- crystallisable fragment
describe the constant region of immunoglobulins
- bottom half of the Y arms and the entire base of the Y
- light chain (CL) = 110 aa
- heavy chain (CH) = 330-440 aa
what is the hinge region of immunoglobulins?
- the region where the arms of the antibody from a Y
- there’s some flexibility in the molecule at this point which is important for funtion (except IgM and IgE)
describe the “domains” of immunoglobulins
- the domains are folded into globular regions, each of which contains an intra-chain disulfide bond, resulting in the characterisitic Ig fold
- there are 12 domains
describe “oligosaccarides” in terms of immunoglobulins
carbohydrates are attached (N-linked) to the CH2 domain in most immunoglobulins
Edelman and Porter won the nobel prize for determining the structure of antibodies using which two enzymes?
papain and pepsin
what is the function of papain?
enzyme present in papaya that is known to cleave the Fc region from the Fab region of an immunoglobulin
how does papain carryout its function? what is the result?
cleaves Ig just below the hinge, resulting in 3 fragments (2 Fab and 1 Fc)
what determines the specificiy of the Fab region?
the VH and VL domains
what is the function of pepsin?
enzyme produced in the human stomach that cleaves the entire Fc region into small fragments (5 + product) up to the hinge
what is the remaining Ig fragment reffered to as after digestion with pepsin?
F(ab’)2 region
what distinguishes the different anitbody classes?
the heavy chains
how many types of Ig classes are there and what are they?
5: IgG, IgA, IgM, IgD, IgE
how to the Ig classes differ?
size, charge, aa composition, carbohydrate content, and thus, fucntion
what is the corresponding heavy chain and serum concentration for IgG type immunoglobulins?
gamma, ~9.0mg/mL (80%)
what is the corresponding heavy chain and serum concentration for IgA type immunoglobulins?
alpha, ~3.0 mg/mL (10-15%)
what is the corresponding heavy chain and serum concentration for IgM type immunoglobulins?
mu, ~1.5 mg/mL (5-10%)
what is the corresponding heavy chain and serum concentration for IgD type immunoglobulins?
delta, ~0.03 mg/mL (0.2%)
what is the corresponding heavy chain and serum concentration for IgE type immunoglobulins?
epsilon, ~0.0003 mg/mL (<0.001%)
following an immune response, what is the domminant Ig produced?
IgG
what are the main characterisitics of IgG? (4)
- major microbial activity in plasma and extravascular fluids
- activates complement when IgG is bound to an antigen
- binds Fc receptors on a varitey of cells
- crosses placenta, acting as a form of natural passive immunity for the fetus
how many human IgG subclasses are there and what are they?
IgG1, IgG2, IgG3, IgG4
can IgG1 cross the placenta?
is IgG a good complement activator?
what is the opsonization for IgG1?
can cross placenta
++ (good compliment activator)
high affinity
can IgG2 cross the placenta?
what is the complement activator for IgG2?
what is the opsonization for IgG2?
cannot cross the placenta
+++
low affinity
can IgG3 cross the placenta?
what is the complement activator for IgG3?
what is the opsonization for IgG3?
can cross placenta
+
high affinity
can IgG4 cross the placenta?
what is the complement activator for IgG4?
what is the opsonization for IgG4?
can cross placenta
- (can’t activate complement)
low affinity
where is IgA typically found?
in mucous secretions
in mucous secretions, IgA exists as:
-a dimer with a joining chain and a secretory component
(looks like a bone because have two Y’s linked together at the tail)
-however, predominantly a monomeric structure
how many subclasses of IgA are there and what are they?
IgA1 and IgA2
describe the structure of IgA1
T-shaped structure due to its hinge region
where is IgA1 found?
in the aerodigestive tract
describe the structure of IgA2
y-shaped structure due to its lack of a hinge region and the fact that the light chain is non covalently bound to the heavy chain
where is IgA2 found?
in the large bowel
what are the 3 main characterisitics of IgA?
- major microbial activity in secretions
- present in colostrum (first milk after parturition)
- does not activate classical complement pathway
what represents the principal antibody class at mucosal sites?
IgA
what is the first immunoglobulin to appear in the normal immune response?
IgM
in human serum, IgM exists as:
a pentamer: 5 IgM monomers joined by sulfide bonds and a J-chain
in addition to in the serum, IgM is a:
monomer - B-cell surface antigen receptor
what are 5 key characteristics of IgM?
- largely confined to the bloodstream
- natural antibodies (for e.g., anti-blood type A)
- good agglutinator
- excellent complement activator after antigen binding
- lacks a hinge region
IgD is largely confied to:
the membrane of mature B-cells together with membrane IgM
is IgD expressed on immature B-cells?
no
does IgD have a hinge region?
yes
what are the known biological effects of IgD?
there are none
compare the structure of IgE and IgG
IgE is slightly larger and more rigid than IgG but looks the same (typical Y)
what are 4 key characteristics of IgE?
- no hinge region
- binds to Fc region receptors on mast cells and basophils
- classical antibody anaphylaxis and certain parasitic infections
- normally very low levels in human serum
how many epsilon constant domains does IgE have?
4
overview: which Ig subtypes lack a hinge region?
IgM, IgE, IgA2
define “isotypes”
pheotypic variations in the constant regions (antigenic determinants) that define each class of Ig
what are the heavy and light chain isotypes?
heavy (found in the Fc region): γ, α, μ, δ, ε
light (found in the constant region): κ, λ
define “allotype”
-slight differences in the aa sequences of heavy/light chains of different individuals
why do allotypes occur?
bc allelic variation occurs in the constant heavy and light chain regions of a specific isotype
where can allotypes be found?
IgG, IgA2 heavy chains and κ light chains
what are allotypes used for clinically?
paternity testing
define “idiotypes”
changes in the variable region (which is antigenic)
why do idiotypes occur?
changes in the variable region result in the recognition of specific antigenic epitopes
antigens often have multiple epitopes, each requiring a different antibody for binding, true or false? why?
true
is any given plasma cell that produces antibodies capable of producing more than one specific type of antibody?
no - only one that can therby target only one specific epitope
do polyclonal or monoclonal antibodies lead to a more effective immune response?
polyclonal
why do researchers often work with monoclonal antibodies, despite them being less effective?
better for controlling their experiments
describe Kohler and Milstein’s experiment for discovering the principle for monoclonal antibody production
- they developped a technique to produce unlimited quantities of identical antibody molecules; by fusing a mutant myeloma cell line (can’t produce antibodies) with spleen cells from a mouse that had been immunized with antigen (produces antibodies), creating a hybridoma cell line
describe the hybridoma cells from the Kohler and Milstein experiment
- these cells aquire the antibody-producing ability of the plasma cells (mouse) and exaggerated longevity of the mutant myeloma cells (human)
- a culture of these cells generate a pool of monoclonal antibodies that can be used clinically
does the mutant myeloma cell line produce antibodies that are unable to grow on HAT medium? why or why not?
no - due to mutations in HGPRT and tyrosine kinase enzymes
how might a physician test for multiple myeloma? why?
urine test - to measure the presence of light chains (reffered to as bence-jones proteins) bc this pathology results in excessive amounts of light chains in the blood, which are filtered as waste in the kidneys
