Extra Cards (Not needed) Flashcards
Monoclonal and Polyclonal antibodies differences
Monoclonal:
homogeneous population of antibodies
made by a single clone of plasma B cells
highly specific for a single epitope on an antigen
Production is expensive
Less cross reactivity
Polyclonal:
heterogeneous mixture of antibodies
made by different B cell plasma clones
bind to the same antigen but may attach to different epitopes of that antigen
Production is inexpensive
Greater cross reactivity
Describe the prozone effect and effect
A phenomenon in which visible agglutination or precipitation does not occur in mixtures of specific antigen and antibody because of antibody excess. This can result in false negative recations.
Lack of agglutination at high antibody concentrations = prozone effect
False negative reactions
Describe Hook Effect
Antigen excess occurs when antigen is present in such high levels that it limits the antigen-antibody crosslinking, resulting in the formation of smaller immune complexes causing immunoassays to underestimate high concentrations of protein.
Agglutination vs percipitation
Agglutination: antigenic particle has antigens which bind to antibodies
Precipitation:
Free antigens bind to free bound antibodies to form a lattice structure
Direct Agglutination vs Passive Agglutination
and example of Direct agglutination and tube agglutination
Direct:
antigens are found naturally on a particle (eg RBC, bacteria)
-Tube agglutination, slide
- Coombs Test (indirect and direct method using overall direct agglutination principles)
-CAT
Passive:
employs carrier particles that are coated with soluble antigens (eg Latex, charcoal)
Widal Test (direct) Brucella serum antigen test (tube)
Haemagglutination – Column Agglutination Technology (CAT) principle
The microtubes contain a gel matrix to trap agglutinates.
- In a positive reaction, as the card is centrifuged plasma and/or RBCs come into contact with the antibody and agglutinated RBCs are trapped in the gel particales.
- In a negative reaction non-agglutinated RBCs are allowed to pass through the column
Viral Haemagglutination methods
Many viruses have the ability to agglutinate RBCs without antigen–antibody reactions
used to detect antibodies in patient’s sera that
neutralize the agglutinating viruses (Viral hemagglutination inhibition test)
Process of Viral hemagglutination inhibition test
and what is the titration
Patient’s serum is first incubated with a viral preparation.
- Then RBCs that the virus is known to agglutinate are added to the mixture.
- If antibody is present, this will combine with viral particles and prevent agglutination.
- Lack of agglutination indicates presence of antibody in patient’s serum.
Titration - The highest dilution of serum (Ab) that prevents hemagglutination is called the HAI titer of the serum.
Complement Based Immunoassay principle and titration
With some antigen-antibody systems no visible reaction occurs.
- The complement fixation test can be used to detect the presence of either specific antibody or specific antigen in a patient’s serum, based on whether complement fixation occurs.
- Indicator system = red blood cells plus their haemolysin (antibody).
- Results are reported as the highest serum dilution not “fixing” complement
Draw the process of complement fixation
Lecture Slide
Neg, Pos, Substrate Blank, Standards purpose ELISA
Negative control – has no analyte
Positive control – contains the analyte
Standards – contain the analyte of known concentration
Substrate blank – Necessary to evaluate the substrate reaction.
- This well does not receive any sample or detector antibodies.
- The blank wells also control for any variation,
- Expected values for the blank are quite low, approaching zero.
Example of
- lateral flow test
- Direct Agglutination
- Passive Agglutination
- Indirect
- Pregancy test or malaria test for parasite detection
- Widal Test
- Treponema Pallidum
- Coombs test
