Assays

Prozone effect

All agglutination assays are subject to misinterpretation due to high antibody concentration relative to antigen resulting in small cross-linking complex; what is this known as? Hint: represents "A" in the image.

Enzyme-linked immunosorbent assay (ELISA)

An enzyme immunoassay performed in a microtiter plate so that color formation in the fluid phase can be *measured in a spectrophotometer*.

Radioimmunoassay (RIA)

An immunoassay in which the detection antibody is linked to a *radioisotope instead of an enzyme*. The reaction between antigen and antibody is *measured by scintillation counting*.

Direct assay

Antigen directly adsorbed onto the solid phase is detected by a labeled *antibody specific for the antigen*.Variation of solid phased immunoassay denoted by #2 above.

Indirect assay

Antigen directly adsorbed onto the solid phase is first incubated with patient serum, and then with a labeled secondary *antibody specific for human immunoglobulin*. Variation of solid phased immunoassay denoted by #3 above.

p24, gp41, and gp120/160 antibodies

CDC criteria for a positive HIV test the patient serum must contain at least two of what?

Enzyme immunoassay (EIA)

Detection antibody is tagged with an enzyme that cleaves a colorless substrate to yield a colored product indicating a positive reactions (as seen in the image); also a similar method used as an initial screening test for HIV (different antibody/antigen).

Immunofluorescence assays

Detects microbes in clinical specimens or autoantibodies bound to self tissues; uses fluorochromes that emit colored light when exposed to UV light.

Western blot (immunoblot)

Identifies an antigen based on its *molecular weight*; PAGE, transfer, block protein binding sites, incubate mb w/patient antiserum, incubate mb w/enzyme-labeled secondary antibody specific for human immunoglobulin (indirect), add colorless substrate, observe for color development of immunoreactive bands (can also be detected via chimiluminescent substrate or radiolabeled secondary antibody).

Antiglobulins

IgGs can overcome RBC zeta potential using *animals antibodies* (anit-IgG) to bind to IgG antibodies, these are also also known as what? (Hint: antibodies to antibodies)

Indeterminate western blot

In the image above, what is the determination of the western blot for patient A?

Possitive western blot

In the image above, what is the determination of the western blot for patient B?

Crosslinking

It is extremely important for both the antibody and the antigen have two or more epitopes/valences so that what can lead to agglutination?

Preciptitation reactions

Ouchterlony test (or immuno-double diffusion technique), Radial immunodiffusion (RID) test, and Immunoelectrophoresis are all examples of what reaction involving *soluble particles*?

Ouchterlony test (or immuno-double diffusion technique)

Precipitation reaction using well cut in an agar gel, the reactants diffuse in the gel and form gradients of concentration, with highest concentrations closest to the well. Between the two wells, the reacting antigen and antibodies will be present at optimal proportions (equivalence zones) for formation of visible precipitate.

Immunoelectrophoresis

Precipitation test that combines electrophoresis with immunodiffusion where antigens are separated in an agar gel by applying electrical charge. Often used to *characterize human serum proteins*.

2 Part testing protocol for HIV infection status

Screening test of enzyme immunoassay (EIA) confirmed by a western blot/HIV-1 IFA.

Fluorescence-activated Cell Sorting (FACS)

Tag unique markers of different cell populations with different fluorochromes, used to identify tissue lineage, maturation stage, or activation status of a cell; cells are passed through a flow cytometer and deflected with a laser for sorting based on intensity of the fluorescent signal.

Epitopes

The particles must have two or more ___________(antigenic determinants) that can act as antibody binding sites before cross-linking (and thus agglutination) will occur.

Solid phase immunoassays

These use solid supports (plastic, nitrocellulose, polyvinylidene, fluoride membranes, spherical beads, ect.) to immobilize the antigen and antibody reactants; *most sensitive and widely used*; include EIA, ELISA, RIA, & Western blot (immunoblot).

Radial immunodiffusion (RID) test

This precipitation test utilizes a homogenous mixture of antibody/gel with cut wells of antigen; results are used to find the *quantitative concentration of antigen* in serum because the precipitin ring is directly proportional to the concentration of antigen in the well.

Negative western blot

What does a western blot with no bands whatsoever mean?

Carbohydrates

What does the anti-A/B sera react to in blood?

Direct immunofluorescence (DIF)

What is #1 in the image above?

Indirect immunofluorescence (IFA)

What is #2 in the image above?

Equivalence zone

What is denoted by "B" in the image above?

Western blot or HIV-1 IFA

What is the confirmatory second test for HIV?

1:32

What is the titer of the above serum assay for anti-Rh factor?

Zeta potential

What repulsive force prevents normal RBCs from agglutinating in the presents of IgG?

Agglutination

When *insoluble*, particulate antigens are cross-linked by antibodies, resulting in clumping of the particles.

X-ray film

When western blots are performed using radiolabeled or chemiluminescent antibodies, the bands are visualized by exposure to what?

Fab fragments

Which *cannot* agglutinate antigens?

IgM

Which antibody type, due to its large size, is likely used to determine the blood typing in the agglutination reactions above, despite the zeta potential between the RBCs?

Heterophile antibody test for infectious mono

Which assay utilizes RBCs from animals to test for specific human antibodies?

A+

Which blood type is #1?

B-

Which blood type is #2?

AB+

Which blood type is #3?

O-

Which blood type is #4?

B

Which in the image above is a positive agglutination reaction?

Indirect Coombs test

Which test evaluates *maternal serum* for circulating anti-Rh antibodies?

Direct Coombs test

Which test evaluates a *fetus' RBCs* for bound maternal anti-Rh antibodies?

EIA & western blot/HIV-1 IFA

Which tests are used for the 2 Part testing protocol for HIV infection status?

Double-antibody, sandwich, two-site capture assay

Which variation of solid phased immunoassay is #1 an example of?