Practice #2 Flashcards
Ag binding site of an Ab determines the _________ of the Ab
Idiotype
Constant region of the Ab determines the __________ of the Ab
Isotype
IgA
Monomeric, dimeric or trimeric Found mainly along mucous membranes but also on the blood in small amount
IgD
Monomeric Antigen receptor on naive B cells Role in activation of basophils, mast cells
IgE
Monomeric Role in allergic reactions and antiparasitic immunity
IgG
Monomeric Mainly in the blood Can cross the placenta
IgM
Pentameric or monomeric as BCR Primary antibody response
What are the different Ig concentrations in the serum
IgG 12mg/ml IgA 2.1 IgM 1.5 IgD 0.04 IgE 0.00003
Ab-Ag possible bonds
- Hydrogen 2. Ionic 3. Van der Waals 4. Hydrophobic interactions NOT covalent
Affinity of Ab-Ag binding
The strength of the Ab-Ag bonds Ex IgG after affinity maturation has higheraffinity for the Ag then IgM
Avidity of Ab-Ag binding
The overall strength of binding is higher Depends on the number of possible bonds (valency) or if it can bind more then 1 epitope on the antigen (polyvalent) Ex IgM is a pentamer with 10 Ag binding sites that can bind different (similar) epitopes on the antigen hence its avidity is higher then that of IgG
Single radial immunodiffusion assay (MANCINI)
- Plate of agarose gel with polyclonal antibodies 2. Holes inside the gel contains blood serum from the pt 3. Incubation 4. Ag from the serum diffuse into the gel and form immune complexes in the equilibrium point between the Ag:Ab and the diffusion will stop 5. We can measure the concentration of complement proteins in the serum that way (linear relationship)
Two dimentional single immunodiffusion assay (MANCINI)
The same process like the single radial MANCINI just now we check not only the Ag concentration but also the Ab concentration. The set up is exectly the opposite. Antigens in the agar plate and Ab from the serum will diffuse out of the hole.
Methods for detection of the antibody as a protein
- MANCINI 2. Electrophoresis 3. Precipitation Agglutination tests 4. Immunofixation
Serum (zone) electrophoresis
Detection of the constitution of blood plasma proteins (globulins and albumin) Sepration by electrophoresis and dye by Coomassie blue From negative to positive: gamma globulins beta globulins alpha2 globulins alpha1 globulins (fewer in num) albumin (highest in num)
Densitogram
Normal
Densitogram
Chronic liver disease
(hepatitis)
Densitogram
Multiplex myeloma
Densitogram
Polyclonal gammopathy
(normal immune rsponse)
What is the meaninng of the M spike in MM densitogram
Production of multiple monoclonal antibodies
What are the indications for electrophoresis?
- to detect paraproteins or misfolded proteins
- to detect protein deficiencies
- to measure relative conc of serum proteins
Esterase reaction
With and without NaF
- Synthetic substrate is entered to a WBC
- Leukocyte esterase converts it to naphtol
- Insertion of diazonium
- Diazonium reacts with naphtol to form a brown product
In the presence of NaF, leukocyte esterase will be inhibited in megakaryocytes, platelets, plasma cells and monocytes but not in lymmphocytes and granulocytes
Immune complex definition
Soluble Ag-Ab complex
Methods used to measure the conc of immune complexes
- Turbidimetry: intensity of transmitted light
sensitivity threshold 50micog/ml
- Nephelometry: intensity of scattered light
sensitivity threshold 1microg/ml
Precipitate
Insoluble aggregates of Abs and Ags
when there is equilibrium between Ab and Ag
Precipitation depends on
- valency
- temperature
- salt conc
- pH
- time
Immunofixation assay
For the detection of a particular Ab isotype
The sample can be any body fluid
placed on a plate with specific antibodies for different Ab isotypes
The complex is fixed and stained
Immunofixation test in MM will find
IgG kappa or lambda light chains in the urine
Bence Jones proteins
Agglutination
Clumping that occurs due to binding of Ab and Ag of corpuscular nature
Ex: RBC, WBC, bacteria, latex
Agglutinins
Ab causing agglutination mainly pentameric IgM
Active agglutination
- direct
- indirect
The particle contributes to the reaction with its own Ag
- Direct: binding of 1Ab is enough for agglutination
- Indirect: secondart antibodies are needed for agglutination
Passive agglutination
The particle serves as a carrier for the Ag or hapten to which the Ab binds
Positive agglutination reaction
Equivalence between Ag-Ab
slow sedimentation
Antibody titer
The highest degree of dilution where hemagglutination still occurs
- no infection
- acute infection
- repeated infection
- immune suppression
- defense
- asymptomatioc infection
Passive agglutination tests
- Antigen is attached to latex beads and the presence of Ab is detected
- Ab are attached to latex beads and the presence of Ag is detected
Applications: test for meningitis capsular Ag, RF
