B Cell Development and Application of Antibodies

Question 1

What are the primary lymphoid organs for T and B cells?

Answer 1

-T cells :thymus

-B cells: the red bone marrow
(primates and rodents)
* the ileocecal Peyer’s patches (ruminants, pigs, horses
and dogs)
*bursa of Fabricius (birds).

Question 2

what happens to lymphocytes in the primary lymphoid organs?

Answer 2

-lymphocytes develop and express their antigen-specific receptors, and V(D)J recombination takes place here

Question 3

What are the secondary lymphoid organs?

Answer 3

lymph nodes, spleen, MALT, Bone marrow

Question 4

What are the steps involved in the development of B lymphocytes?

Answer 4

start primary lymphoid organs (antigent independent):

1.Pro-B cell
2. Pre-B cell
3.Immature B cell
4. Mature B cell

then move on to the SLO (ARE ANTIGENT DEPENDENT)

5. memory B cells
   6.Plasma cells

Question 5

describe the Pro-B cell stage

Answer 5

B lymphocytes arise from hematopoietic stem cells that commit to the B cell lineage.

earliest distinguishable cell in the B cell lineage

have undergone D-J segment heavy chain rearrangement but have NOT produced immunoglobin (Ig) for expression on their cell surface

Question 6

Describe the Pre-B cell stage

Answer 6

cell makes μ HEAVY CHAINS BUT NO LIGHT CHAINS.

have undergone COMPLETE V-D-J segment HEAVY CHAIN rearrangement.

This rearranged V-D-J unit is put next to the Cμ gene, and the pre-B cell produces a μ heavy chain

μ heavy chain is paired with SURROGATE light chain to form a molecule that can be expressed at the cell surface

Ig-ALPHA and Ig-BETA are obtained on this stage as well

Question 7

what is a surrogate light chain, which is seen in Pre-B cells? and what is therefore the Pre-BRC?

Answer 7

product of two non-rearranging genes

μ heavy chain PLUS surrogates light chain are known as Pre-BCR

Question 8

plasma cells created first in the primary response will secrete _________, UNTIL TJE IMMUNE SYSTEM PROCEEDS and the TH CELLS BECOME AVAILABLE FOR CLASS SWITCHING

Answer 8

IgM.

Question 9

_______ are necessary to carry out class switching , Where class switching prodigy will yield______

Answer 9

Th cells

plasma B cells (G,A or E) AND memory cells (G,A or E) that secrete their immunoglobulins on the cell surface.

Question 10

describe the Immature B cells stage

Answer 10

have undergone a
COMPLETE V-J LIGHT CHAIN rearrangement

actual light chains are paired with μ heavy chain

EXPRESS IgM (with two FULL light chains, and two FULL left chains)

Question 11

What are the possible outcomes of an immature B cell the tissue exposed to an antigen?

Answer 11

1. Deletion-death of that cell by apoptosis
2. anergy- or the cell
   is inactivated
3. receptor editing-the cell’s Ig L chain genes undergo rearrangement using unrearranged V or J elements (generate new specificity for IgM)

Question 12

Describe the mature cell stage

Answer 12

-Immature B cells develop into mature B cells that have both membrane IgM
and membrane IgD

-mature B cells migrate to the 2 lymphoid organ (spleen, lymph nodes, peers patch, tonsil, appendix) in order to…

***seek out FOREIGN ANTIGEN to produce PLASMA CELLS and MEMORY CELLS

Question 13

Describe plasma cells

Answer 13

-the interaction of an
antigen with a mature B cell results in activation and PROLIFERATION

-During the early stages of primary immune response, plasma cells secrete
antibody molecules of the IgM class UNTIL TH CELLS ARRICE AND SEND class switching and somatic hypermutation signals to B cells through cytokines

***the light chain DOES NOT UNDERGO class switching, they only UNDERGO somatic hypermutation

Question 14

class switching keeps the original antigen specificity, however, somatic hypermutation (in Vh and VL domains) produces B cells whose Ig variable region genes can synthesize antibodies with HIGHER AFFINITIES

Question 15

Somatic hypermutation in B cells occur _____ receiving Th cell cytokine help.

Answer 15

after

Question 16

What is the next step that follows somatic hypermutation?

Answer 16

selection of useful and high affinity B cells:

-dendritic cells present B cells with antigens in THE GERMINAL CENTERS, and those that effectively bind, are selected to survive

-as each antigen is cleared (and less and less are available), those B cells with antibody mutated to have HIGHER affinity will be able to bind antigen and survive.

-Those B cells that do not bind to an antigen will die by apoptosis, and those with lower affinity also, will die due to being unable to complete their job

Question 17

Somatic mutation and class switching occur ONLY IN _____

Answer 17

B cells and NOTTTT in T cells

Question 18

what are memory B cells?

Answer 18

-long- lived B cells that appear during clonal expansion

-DO NOT secrete Ig and remain dormant until SUBSEQUENT exposure of antigen presents itself

-will lead to a larger and faster immune response

Question 19

what are happens in each part of the B cell development?

Answer 19

Question 20

What are B cell receptors?

Answer 20

the complex of membrane Ig along with Ig-alpha and Ig-beta
heterodimer is referred to as the B cell receptor (BCR)

Question 21

what are the three clinical and experimental applications of antibodies?

Answer 21

I.diagnostic
2. therapeutic
3.research

Question 22

What type of antibodies are used diagnostic, therapeutic, or research? and how are they obtained?

Answer 22

Polyconal and monoclonal antibodies

polyclonal (serum) antibodies: antibodies prepared from serum of animals that are 99% purified, and then used for therapy purposes in animals and in humans

Monoclonal antibodies: Immortal “hybridoma” antibody cells that are com e from one particular B cell that was placed on a dish, and immortalized by combining it with myeloma cells to be able to produce ONE antigen binding immunogen therapy
-obtained from mice specifically

Question 23

How can therapy use mouse based monoclonal antibodies in dogs?

Answer 23

-They are modified in order to prevent the anti mouse igG antibodies.

chimeric: have mouse variable regions but have been engineered to have dog constant regions.

canonized: CDR regions of the antigen-
specific mouse antibody “grafted” into a dog immunoglobulin sequence.

canine:whole molecule is dog Ig

Question 24

What are immunoassays? and what are some examples of theses?

Answer 24

laboratory tests that use the immune system’s response to detect and measure specific substances

example:

-Enzyme Linked mmunosorbent Assay (ELISA)

-Immunofluorescence Assay (IFA)

-Radioimmunoassay

Question 25

what is Enzyme Linked Immunosorbent Assay (ELISA)?

Answer 25

-ANTIGEN ANTIBODY reaction measuring magnitude of antibody isotype response against an antigen, pathogen, or vaccine by determining specific titers in serum and other biological fluids.

The basic principle of an ELISA is to use an enzyme to detect the binding of antibody with antigen. An enzyme is coupled to a specific antibody for use as a detector.

Question 26

what are the most common ELISA types?

Answer 26

INDIRECT ELISA(to titrate anti-rabies antibodies):

-antigen coats plate, blocking of unbound sites after,followed by antibody (primary-against canine rabies) and antibody conjugate (secondary antibody- anti-isotype rabies) introduction, and lastly adding a stopped solution.
-SECONDARY ANTIBODY HAS ENZYME LNKED
COLOR MEASURES REACTION
-The antibody titer is
the reciprocal of the last antiserum dilution giving mean OD values
above the cut-off level, which will be twice the mean OD value of
the negative control wells.
-Therefore, a serum antibody titer simply is
the last dilution of the serum that gives a positive result. The dilutions
after that last dilution give negative results.

DIRECT ELISA:

-Same steps as indirect BUT AN ENZYME IS LINKED TO THE PRIMARY antibody before being introduced to the coating section, INSTEAD of the secondary antibody having the enzyme

SANDWICH ELISA:

-ANTIBODY FOR SPECIFIC ANTIGEN is coated to the wall
-The sample is then added to the
wells and any antigen that might be present in the sample is allowed to
bind to the antibody.
-Bound antigen is detected by the addition of a
secondary enzyme-coupled antibody specific for a different epitope
(antigenic determinant) on the same antigen.

**direct and indirect use spectrometer for measures
**sandwich uses OD obtained

Question 27

how is lateral flow test done for pregnancy testing?

Answer 27

• Placenta yields RELAXIN, therefore:
• anti-relaxin Monoclonal antibodies are used (COLOIDAL GOLD) to test for pregnancy

-control is present that binds to other proteins/antibodies

-SANDWICH because it is between two antibodies, but NOT ELISA because it is NOT ENZYME BASED

** same concept is seen in infectious diagnosis, EXCEPT the Mab will look for SPIKE PROTEIN

Question 28

What is the mechanism behind 4DX snap test?

Answer 28

ELISA TECHNOLOGY

• screens for LEAH
  -Lyme (antiboy)
  -Ehrilichiosis (antibody)
  -Anaplasmosis (antibody)
  -heartworm (antigen)

**if antibody present, does not necessarily mean that infected (had infection before), only if antigen present

Question 29

What is immunofluorescence Assay? (IFA) and what is indirect and direct respectively?

Answer 29

Immunofluorescence assay is a powerful technique that utilizes antibodies labeled with fluorescent dyes to detect specific target antigens.

Direct IFA: In a direct immunofluorescence assay, a fluorescent-labeled antibody reacts directly with its target antigen.

Indirect IFA: In an indirect immunofluorescence assay, antigen is first detected using a specific antibody which is not labeled with a fluorescent dye. Any binding of the specific antibody to the antigen is then detected using a fluorescent-labeled secondary anti-antibody. In IFA, the secondary antibody is covalently conjugated to a fluorescent dye.