In vivo an in vitro Antibody use Flashcards
What is the FcRn?
neonatal Fc receptor, binding on the Fc region of the maternal Ab
Function: Transfer Ab into the fetus, and also cross the epithelial cells of the intestine to the blood (breastfeeding)
What is passive immunity?
Immunization by delivering preformed antibodies
from one person to the other
-no long-lived B-cells
-no memory cells -> inhibit memory cell formation
-Short term: IgG last for 2 weeks
-used for immune deficiency (children), Toxins or venoms (life-threatening), or pathogens that kill fast
Why is is the Rh factor dangerous?
1st pregnancy: Blood transfer during delivery or pregnancy -> exposure to fetus Rh antigen on RBC -> formation of maternal Ab and memory cells against Rh
2nd pregnancy: maternal Ab circulates into fetus bloodstream -> causes destruction of fetal RBC by Ab binding and complement
How can Hemolytic disease of Newborns due to the Rh factor be treated?
-intrauterine blood exchange transfusion
-UV light decreases toxic RBC fragments
-use anti-Rh Ab to prevent exposure to Rh-specific B-cells -> no Ab formation, no memory cells
Why are sometimes antibody fragments created in the lab, instead of the complete Ab?
Because the Fc region of the Ab binds to the Fc receptor of immune cells (macrophages, NK cells, neutrophils) and causes inflammation
-> sometimes we just want the Ab to bind a specific enzyme in its active site -> so the Ab is fragmented and only the Fab or F/ab)2 region is used
Difference between Fab and F(ab)2:
Fab consists of 2 different chains a and b (one heavy, one light)
Fab has 1x heavy and light chain binding site
F(ab)2 has 2x heavy and light chain binding sites -> Agglutination, collecting antigens with a “net”
How can Fab proteins be made more stable?
Genetically engineered (scFv) single chain Fv fragments: connecting the heavy and light chain together with binding regions within the same protein
How can engineered Ab fragments be used in diseases?
Chimeric Antigen receptors
create Ab fragments against tumor-specific antigens (f.e. CD19 on B-cell leukemia) on cancer cells to make it a target for T-cells -> activation of a killing cascade
-not really a T-cell receptor, no CD28 - CD80 involved, to alert T-cells to cancer cells
How are antibodies created in the lab?
With antigens given to mice (polyclonal or monoclonal)
adjuvant (f.e. PAMPs) together with Ag needed bc just the protein sometimes doesn’t cause a strong immune response (with cytokines, memory cells)
What are polyclonal Antibodies?
Pro and Cons
When exposing a mouse to an antigen, different B-cells bind to different epitopes of that same antigen -> formation of different Ab -> after a few weeks all the Ab are isolated and purified -> now you have polyclonal Ab
Pro: different Ab will be able to Agglutinate
Cons:
-not specific to inhibit an enzyme (Toxins) on the specific active site
-cross-reactivity
How are monoclonal Antibodies made?
Expose the mouse with antigens and adjuvant -> stimulate an immune response -> instead of isolating the Antibodies, get the B-cells i.e. from the spleen -> hybridize with myeloma cells to produce a high number of antibodies -> dilute in single wells to a single B-cell that produce one specific antibody -> test the single antibodies and find out which one actually inhibits your target
How can mouse-created antibodies be used in humans?
-Humanize the antibodies: replace the mouse Fc region with the human Fc region and keep the mouse Fab region or just the binding site in the Fab region
-Immunotoxin: replace the Fc region with a toxin molecule (how does that Ab bind to the target without Fc region??)
for detection: Conjugation
-with Biotin/enzymes (ELISA), Fluorescent dyes (immunofluorescence or flow cytometry), synthetic beads (immunoprecipitation)
How are modified Ab used in blocking anti-inflammation?
-Blocking of CTLA-4 (inhibits T-cell activation after a few days -> we want to keep the T-cell active in case of cancer)
-APC express inhibitory receptors -> cancer cells mimic that -> blocking of inhibitory receptors
-if you want to kill: conjugate the antibody with a toxin, the toxin will attack the cancer cell
or use the Fc region to bind NK-cells that bind to modified Ab which binds to the target cell -> NK cell kills the target
Modified Ab to target human cancer cells activate which type of innate immune cells?
NK-cells
6 ways how Ab kills:
-Neutralization (block)
-Agglutination
-Opsonization (Macrophages)
-Complement activation
for human cells (they are large)
-Antigen-dependent-cytotoxicity (cytotoxic with NK-cells)
-ADDG (Degranulation: Basophils, Mast cells, Neutrophiles -> worms)
Use of monoclonal Antibodies against tumor cells:
mAb against CD20 B-cell marker
OR
create anti-idiotype mAb binding to the Fab region of B-lymphoma Ab
1. take B-lymphoma and fuse it with Myeloma and select for secreted Antibodies from that B-lymphoma
2. Put the Ab into a mouse, which will form antibodies against that B-lymphoma-Ab in the spleen -> fuse with mouse Myeloma and get the specific Ab that inactivates the B-lymphoma
3. inject into a patient
