Flashcards in Quantitative Assays of Immunity Deck (70):
What type of forces hold Ag and Ab together?
4. van der Wals
What is an ionic force?
attraction between opposite charged groups like O- in carboxylic acid (aspartic acid) with hydrogen in amino groups (lysine)
What three AA side chains form hydrophobic interactions?
Leucine, isoleucine, valine
When leucine, isoleucine and valine interact with each other what happens?
They squeeze out water surrounding the reactants so water is order when it is around Ab or Ag and is disordered when excluded from the Ag-Ab interface
The strength of the interaction between Ab and Ag can be estimated by applying ___________.
Law of Mass action
What measures the strength of an Ag-Ab interaction?
Typical Ab-Ag interactions have dissociation constants in the _______________range.
What is the equation for association constant?
Ka= Kon/Koff = (Ag-Ab/) Ab*Ag
What is the equation for dissociation constant?
Kd= Koff/Kon= Ab*Ag/(Ag-Ab)
Would the Kd be high or low for an Ab-Ag interaction with high affinity?
Low because there would be more complex than free Ag or Ab
What is the difference between affinity and avidity?
Affinity is the strength of the interaction between ONE combining site of the Ab and ONE Ag epitope.
Avidity is the summation of all interactions between combining sites of the Ab (2 for IgG, 5 for IgM) and all the identical epitopes of Ag
Why is avidity NOT the arithmatic mean of the affinity of a single binding site?
Multivalent binding of Ag to each arm of the Ab confers an enhancing effect because when one combining site dissociates the other binding site is still bound and stabilizes the complex
Avidity is only relevant when _______________________ are being considered.
repetitive antigens on a surface
What is cross-reactivity?
Sometimes an Ab directed toward a specific antigen can bind structurally related but not identical isotopes.
They will bind these epitopes with lower affinity then the EXACT epitope they are specific for.
What is a monoclonal Ab?
An Ab derived from a clonal population of cells so it will have specificity for the same epitope on an antigen as its clones
What is a polyclonal Ab?
mixtures of different Ab that bind to different epitopes of the antigen. Some may bind to overlapping or the same epitope of the antigen.
What is the necessary condition for the precipitation of any soluble Ag?
More than one of its epitopes must interact with a bivalent Ab or polyclonal Ab
When a precipitate is forming a lattice of ________ is formed with increased ____ and decreased _______.
What are the two quantitative ways to measure the cloudiness of a mixture of Ag-Ab?
Can a monovalent antigen for a precipitate?
No because there is no cross-linking effects
Antigens with two or more identical epitopes can be precipitated by_____________./
Divalent Abs directed against the epitope (2 antibodies that bind the same antigenic epitope)
Ag expressing two or more different epitopes can be precipitated by ______________________________.
a mixture of divalent Ab (polyclonal)
What does the amount of immune precipitate depend on?
The Ag/Ab ratio
What is it called when there is excess Ab, so weak or no precipitate forms?
What is it called when there is an antigen excess so no precipitate forms?
Only when Ag and Ab concentrations are ___________________________ does optimal precipitation occur. This is called the ___________________.
equivalent or close to equivalence (# of combining sites is equal to the # of epitopes)
Why would an excess of antigen (post zone) not allow for precipitation?
The antigen will bind monovalently so immune complexes will not form
What is the precipitation reaction useful for studying?
What is tubidimetry?
the measure of the reduction of light intensity as a consequence of absorption, reflection and scattering.
What is nephelometry?
the light that is scattered at a particular angle from the incident light beam
What can the amount of light absorbed or scattered tell us about the substance?
the amount of Ag-Ab complexes formed
What are nephelometry and turbidity used in the clinical lab to quantify?
serum proteins such as:
1. classes of Ig
2. Complement components (C3,C4,C1 inhibitors)
What is the sensitivity of turbidity and nephelometry?
How can sensitivity of precipitation reactions be improved?
low- about 1-5micrograms/ml
Sensitivity can be improved by performing the precipitation in semisolid support like agar
How is immunodiffusion performed in agar?
What is the name of this test?
The Ag and Ab are placed in separate wells and allowed to migrate by diffusion or driven by electric current (immunoelectrophoresis)
When the Ab and Ag interact they form a lattice and a line of precipitate is formed.
This is called the Ouchterlony test.
What is Ouchterlony double diffusion used to identify?
What are the drawbacks of this method?
Identify Ag in the biological fluid.
There is low sensitivity and it is time consuming (72 hours)
What is the radial diffusion assay used for?
How does it work?
It is used to quantify the amount of Ab or Ag.
An agar gel that contains Ab and fill wells with Ag, some with known quantities of Ag others (prob from serum) with unknown quantity.
Use ring diameters formed around the well to plot a standard curve of "diameter vs. amount in well".
The curve can determine the "unknown' amounts of antigen.
(this can also be done in reverse where the agar has antigen and the substance in the well is Ab)
How will the lines on the Ouchterlony test form if the antigens are:
1. the same
2. when they share NO common epitopes
3. when they share some common epitopes
1. it is "identity" and the precipitate will form a V
2. it is "non-identity" and the two test antigens will form a cross pattern
3. It is "partial identity" where the lines will partially cross because some antibodies will be cross-reactive and others won't be
How would agglutiation appear on the bottom of a test tube?
It would be spread out along the walls of the test tube (vs. a button formed at the bottom when not agglutinated)
When are the two most common scenarios for using an agglutination test?
1. in Ob/Gyn for Rh+ tests
2. Before giving a blood transfusion
What is the necessary condition for agglutination to occur?
The surface of the particle must express multiple epitopes and the Ab combining sites must be aboue to encompass the spatial distance between the two particles
What is the distance between 2 RBCs determined by?
The repulsion force of their negative charges (zeta-potential)
What is the only Ab that can agglutinate RBCs?
IgM because it is a large enough molecule to span the distance between two RBCs.
People with blood type A have what Abs?
RBCs belonging to a person with blood type A can be agglutinated only with the serum harvested from ____________________.
a person belonging to blood group B that makes anti-A
At low dilutions of serum, will agglutination occur?
No because the Ab conc is too low
If a Rh negative woman has children with a Rh+ man, what could occur in the children?
The first child would be fine, but subsequent RH+ children may have hemolytic disease because the mother's serum will make anti-Rh antibodies that are able to cross the placenta
What is used to stop the Rh- mothers serum from attacking Rh+ RBCs in subsequent pregnancies?
At the first pregnancy, the woman is given RhoGAM which suppresses her immune system to not attack the Rh+ blood cells (so she doesnt form antibodies against them)
What is done in the direct Coombs Test to detect the mothers anti-Rh IgG in the baby's blood?
IgG can't agglutinate RBCs on their own.
Anti-human IgG is added to the baby's blood.
If agglutiation occurs then the baby will have hemolytic disease because this means they already have Ab on their RBCs (Anti-Rh Ab)
What is the indirect Coombs test for detecting whether the mother;s anti-Rh+ Ab are attacking the baby's RBCs?
Use the mother's serum and add known Rh+ blood to it. Then add anti-human IgG antibodies. If there is agglutination, then the mother is making Anti-Rh+ Abs
What is immunoblotting (Western Blotting)?
What disease is it used to test for?
It is using electrophoresis to separate Ag mixtures according to molecular size and charge. Then you can incubate them labeled Abs against the Ag of interest.
It is frequently used to check for anti-HIV antibodies.
After the separation of proteins by electrophoresis, they are trasfered from the polyacrylamide gel on the nitrocellulose paper followed by incubation of the paper with _______________________.
labeled Ab directed toward the Ag of interest.
What are the two ways Ab against Ag of interest can be labeled when using Western Immunoblot?
2. Enzymatically - that catalyze colorless substrate to a colored product (used more nowadays)
If anti-HIV antibodies are present on the Western Blot, what does this mean?
The person is HIV positive
In a typical western blot, what is the enzymatically labeled molecule?
The anti-human IgG antibody because it will bind to the Ab of interest, then be converted to colored product to visualize
What do Radioimmunoassays and ELISAs measure?
quantitative measure of the amount of an Ag or Ab in a solution by specific binding with corresponding Ab or Ag and comparing it to a standard dilution
What are some of the Ags and Abs used in ELISA/RIA testing?
HIV Ag or Ab
HepABC Ag or Ab
HTLV I and II Ab
What is non-competitive ELISA?
1. Solid Ag is in the well
2. Serum (with Ab to the Ag) is added
3. Ab binds to Ag if there is Ab present
4. Enzyme-labeled anti-human IgG is added
5. Color development can determine Ab presence
What is a competitive RIA assay?
1. Ab is in the well
2. Add 4 labeled Ag, no unlabeled--> 100% radioactivity
3. Add 4 labeled Ag, 4 unlabeled__>50%
4. Add 4 labeled Ag, 12 unlabeled --> 25%
More unlabeled added, the less labeled will bind and the less radioactivity there will be. You can then generate a standard curve to determine Ag levels in serum
What is a "real life" example of an ELISA?
How does it work?
A pregnancy test.
1. Pee on the stick.
2. If it contains hCG it will complex with colloidal gold-linked mouse anti-hCG
3. bound and unbound anti-hCG will diffuse up the strip to a test region where it complexes with OTHER mouse anti-hCG
4. Unbound Ab continues to diffuse up the strip to the control region where there is goat anti-mouse Ab
5. Colloidal gold molecules form a line in one or both windows (neg and pos)
What is flourescence microscopy used to detect?
the presence of Ag in fixed tissue OR live cell suspension
How do you perform the direct method of floursecence microscopy?
1. Tag Ab with fluorescence
2. It will bind to Ag fixed to a microscope slide
How do you perform indirect method of fluorescence microscopy?
Detect Ab in patient serum know to be specific for an Ag present in a cell smear by fluorescently labeling anti-human IgG
What does the indirect method of fluorescence allow you to visualize?
The places in the cell tissue where the Ag is localized (while also revealing the presence of Ab)
What is flow cytometry?
A system that detects the presence of fluorescent agent on a target and the intensity of the fluorescence.
Can sort cells into different types based on their fluorescence.
How does FACS (fluorescent activated cell sorting) work?
Cells are incubated with different fluorescent labeled Ab then pass through a laser to excite the fluorescence. As they pass the charged plates, they are sorted into different reservoirs based on the type of antibody
How are monoclonal Ab produced in a lab?
1. Mice are immunized with AgX
2. The cells are fused with "immortalized" aka tumor cells from a mouse myeloma
3. Screen for anti-Agx antibodies
4. Allow differentiation of clones
What is a problem with how monoclonal Ab are created now?
They use mice cells so they make mice Ab/ This may cause serum sickness in humans
A new approach is allowing the engineering of antibodies from human Ab genes. What is the first drug developed in this manner?
What is this drug an antibody against?
Humira which is an anti-TNFa antibody
What is phage display to isolate Ab?
1. The Ab variable domain or Fab is attached to the surface of a phage.
2. The phage binds Ag displayed on plastic
3. The phages that bind Ag clonally expand
4. The clone genes can be selected and used to rebuild complete Ab with that variable region