immunodiffusion / lab 5 (LR4)

- a diagnostic laboratory technique used to detect and quantify antigens or antibodies by letting them diffuse naturally through a gel matrix
- as they move through the gel, they meet and bind together
- if they are a specific match, they create an insoluble, visible white line or ring of clumping called a precipitin line
- examples: Ouchterlony Double Diffusion (ODD) & Radial Immunodiffusion (RID / Mancini Method)

- both antigen and antibody diffuse through agarose. a plain agar gel is prepared, and separate wells are cut into it. the antibody goes into one well, and the target antigens go into the surrounding wells. as they diffuse toward one another, they form lines of precipitation
* scientists look at the shape of these lines to determine relationship patterns (identity, non-identity, or partial-identity)

the lines fuse into a continuous, smooth arc, showing the antigens are identical

the lines cross over each other completely, showing the antigens are completely different

a line with a "spur" forms, showing the antigens share some characteristics but are not identical

* core technique used to calculate the specific concentration of an antibody or antigen in a sample
- a specific antibody is mixed evenly right into the gel before it sets
- the sample is placed into a central well and spreads outward radially (in a circle)
- as it spreads, it binds to the embedded antibody and forms a ring
- by measuring the diameter of the ring and comparing it to a standard curve, technicians can determine the exact concentration of proteins like IgG or IgM in the blood

- checking immunity
- determining booster needs
- evaluating autoimmune conditions
- identifying past infections

to identify if a previous vaccine (such as for measles, mumps, rubella, or hepatitis B) or past illness still provides protection

to see if a person needs an extra vaccine dose when immunization records are missing or old

to measure the strength of an abnormal immune reaction against the body's own tissues in conditions like systemic lupus erythematosus (SLE)

to check if someone was infected with specific viruses or bacteria in the past

an active, acute illness; it looks backwards at immune memory or status

* positive reaction = visible white line
* negative reaction = no visible line
- white, curved precipitation lines form between the central antigen well and the antibody wells
- the line is visible when antigen and antibody are at the zone of equivalence
- the highest dilution that still shows a visible line is the antibody titer
- ex: if clear line is visible up to 1:16 but no line is visible at 1:32, the antibody titer is 1:16

* strong reaction = larger ring at higher antibody concentration
* weaker reaction = smaller ring at lower antibody concentration
- white circular precipitation rings form around the wells containing antibody
- the diameter of the ring increases as the antibody concentration increases
- the blank (dilution buffer) should have no ring
- measure the diameter of each ring (in millimiters) using a ruler or calipers
- use the standard ring diameters to create a standard curve and determinethe concentration of the unknown sample

* measure the full outer diameter (mm); do NOT measure only the clear well or the ring thickness
1) pace slide over dark background
2) locate the outer boundary of each white precipitate ring
3) measure straight across its center, from outer edge to outer edge (mm)
4) record all 6 standards and the unknown; the blank should NOT form a ring