Section 1: Agglutination and Precipitation Reactions Flashcards

1
Q

unlabeled immunoassays

A

-agglutination and precipitation were the first immunoassays developed and are based on cross-linking of antibody-antigen complexes
-these reactions are not that sensitive because results are visualized by eye or using optics (turbidometry and nephelometry)

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2
Q

precipitation

A

-cross-linking of a soluble antigen to create an insoluble precipitate

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3
Q

agglutination

A

-cross-linking of particulate antigens to form larger, also visible complexes (bacteria, cells, latex beads)

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4
Q

Affinity

A

-all serological assays (labeled or unlabeled) rely on the affinity, avidity, and specificity of the Ab to the Ag

-the strength of the binding of 1 Fab region with its corresponding Ag

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5
Q

Affinity constant (Ka)

A

(Conc. of Ab-Ag complex)/ (Conc. Ab) (Conc. Ag) = (AbAg)/(Ab)*(Ag) = Ka
-the higher the affinity the more sensitive the reaction

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6
Q

avidity

A

-the number of binding sites times the affinity
-IgG with two binding sites has an avidity of 2 * (Ka)
-IgM with 10 binding sites has an avidity of 10 * (Ka)
* IgM usually has a lower affinity than IgG because the production of IgG (class switching) has occurred after somatic hypermutation events
-Steric hindrance can also play a role in lowering avidity
-the greater those numbers are the lower amount we can detect

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7
Q

equivalence

A

-the the formation of Ag-Ab complexes the largest complexes are formed when Ag epitopes are in equivalence to Ab binding sites

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8
Q

prozone

A

-when precipitation efficiency is decreased because of excess Ab which decreases cross-linking
Prozone: dilute patient serum (can try to dilute patient serum)

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9
Q

Postzone

A

-when precipitation efficiency is decreased because of excess Ag which decreases cross-linking
** both prozone and postzone will appear falsely negative
Postzone: redraw 1-2 weeks after more Ab produced

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10
Q

What is the simplest way to detect Ab-Ag interactions

A

precipitation
* this is accomplished by placing Ag and Ab in different wells of an agar plate, allow time for them to diffuse through the agar, and visualize the precipitation pattern

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11
Q

Advantage of precipitation

A

-easy and cheap

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12
Q

Disadvantages of precipitation

A

-limited sensitivity (20 ug/ml)
-cannot be amplified, either by attaching Ag to a particle or linking of a substance to the Ab to increase visualization

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13
Q

Ouchterlony analysis

A

-double diffusion gel precipitation
-qualitative
-both Ag and Ab diffuse through agar media
-as radial diffusion occurs Ag and Ab reach equivalence a precipitate is formed by cross-linking (lattice structure)
-particularly useful in identifying if unknown Ag is Identical, Partially, Identical, or Nonidentical with a known Ag
-modern uses are limited to rare fungal infection diagnosis (coccidiomycosis) and in some Ab specificity testing in autoimmune diseases

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14
Q

Ouchterlony (Line of identity)

A

-when identical Ags are placed in the wells, a line of identity is formed (ARC)

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15
Q

Ouchterlony (Line of nonidentity)

A

-when completely different Ags are placed in the wells, two crossing lines of precipitate form, line of nonidentity

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16
Q

Ouchterlony (Line of partial identity)

A

-when similar Ags are placed in the wells, a line similar to the line of identity forms with the exception of forming a spur
-the spur always points to the simpler Ag, that is the one with fewer epitopes

17
Q

Radial immunodiffusion (RID)

A

-based on a concept similar, except now there are many wells with decreasing concentrations of Ag as you move left to right across the agar media which already contains Ab
-Quantitative
-more concentrated Ag wells diffuse farther into the media
-conc. can be calculated by measuring the diameter of the precipitate line from the well and producing a standard curve with known Ag concentrations
- RID is used to measure patient IgG, IgM, and IgA levels (the Ab are being measured as Ag because an anti-human Ab is contained in the media, recognizes Fc portion)
-mobilize one antibody into the agar itself, so the agar contains one of the two factors at a constant concentration, then vary or dilute out the patient sample and see where the lines come

18
Q

Two methods to perform RID

A
  1. Fahey method (kinetic method)
  2. Mancini Method method (End-point method)
19
Q

Fahey method (kinetic method)

A

-difusion proceeds for 18 hours
-the diameter is proportional to the Log of the concentration
-plotted on semilog paper (x axis = diameter, y axis= concentration)

20
Q

Mancini method (end-point method)

A

-diffusion proceeds for 48-72 hours
-the square of the diameter is directly proportional to the concentration