Section 3: Labeled Immunoassays

Question 1

Labeled immunoassay

Answer 1

-a marker or label is attached to an antibody or antigen, thus increasing the sensitivity

Question 2

unlabeled immunoassays

Answer 2

-are cheap and easy to perform but limited by their sensitivity (visualization of precip or agglunt.)

Question 3

Radioimmunoassay (RIA)

Answer 3

-was the first labeled immunoassay developed by Rosalyn Yalow in 1959 to detect insulin
- Competitive RIA (patient Ag competes with radiolabeled Ag)
-Ag binds to a fixed amount of Ab
-Concentrations measured at lower levels than possible by eye
-1000 fold increased sensitivity

Question 4

What are some of the various labels and markers that can be used?

Answer 4

-radiolabel (radioactive isotope) (RIA)
-Enzymes (enzyme immunoassay) (EIA/ELISA)
-Fluorescent compounds (FIA)
- Chemiluminescent label
-Colloid particles

Question 5

What are the two types of assays?

Answer 5

1. Heterogeneous Assay
2. Homogeneous Assay

Question 6

Heterogeneous Assay

Answer 6

-requires separation of bound label from a free label (solid phase for facilitating the reaction, followed by a wash)
-use solid phase attachment of Ab or Ag to a microtiter plate, slide, or bead that allows for rapid separation of bound label from free

Question 7

Homogeneous Assay

Answer 7

-does not require separation or washing because the binding of an Ag to Ab directly affects the activity of the label

Question 8

colloid immunoassays

Answer 8

-detected by eye after separation (immunochromatography)
ex: point of care testing, home pregnancy tests

Question 9

Types of heterogeneous immunoassays

Answer 9

1. direct
2. indirect
3. competitive
4. sandwich or capture
5. western blot

Question 10

Direct Immunoassay

Answer 10

-labeled antibody binding to antigen or labeled antigen binding to antibody
(Direct)
-directly labeled targets (ex: primary antibody)

Question 11

Indirect immunoassay

Answer 11

-unlabeled antigen and unlabeled antibody are used in conjunction with a labeled detection (or secondary) antibody

Question 12

Anti-Human immunoglobulin

Answer 12

-key reagent in indirect immunoassay
- can detect all or specific isotypes of Ab (IgM, IgG, IgE)
-various labels can also be used in indirect assays

Question 13

capture immunoassay (sandwich)

Answer 13

-captures antigen between 2 antibodies, usually, one antibody (capture Ab) is bound to a solid-phase medium and the other antibody is labeled (labeled Ab)
EX: immunochromatographic sandwich assay: home pregnancy test
-looking for antigens

Question 14

double capture

Answer 14

-an assay that can test for patient antibodies (West Nile virus)
-was developed to detect IgM preferentially over IgG for the diagnosis of a current infection
-looking for antibodies

Question 15

Why do patients with rheumatoid arthritis produce false positives?

Answer 15

Rheumatoid factor is a human antibody (predominantly IgM) but can be other ones, that respond to the fc portion of human IgG. So if you use that assay to detect IgM and you have human antibodies here, if RF Is there it is going to bind to stuff indiscriminately and nonspecific to the assay

Question 16

Why will capture assays not work?

Answer 16

1. Ag is too small
2. Ag has only one epitope
3. Ag has multiple epitopes that cause steric hindrance of the reaction

Question 17

steric hindrance

Answer 17

when a reaction is inhibited because of the size and or shape of molecules involved in the reaction

Question 18

Competitive Immunoassay

Answer 18

-because less substrate is created when patient Ag or Ab competes with kit reagent, an inverse relationship exists between substrate and amount of patient analysis
-we have a bead or substrate in the assay that acts as the solid phase or substrate. We coat that bead with our antigen and add our antibody complex with our label. If nothing we get a strong inhibited signal. Bead and antibody do not change but patient samples compete with those binding sites and sequester them off. Wash-off will have a decreased signal
-

Question 19

Multiplex (microbead assay)

Answer 19

-microbeads coated with Ag-specific to reflex testing of a positive ANA are used to examine patient samples for multiple antibodies simultaneously
-each bead has its own internal fluorescent signature
-used on cytokines, can detect antibodies or auto nuclear antibodies (ex; part of lupus testing)
-read a combination of colors, for a true read you need not just one color

Question 20

Homogenous assays

Answer 20

-do not require the separation of bound analytes from free ones (easier than heterogeneous)
-also bound to Ag in a homogeneous assay causes a direct change in the visualization compound
-used for small molecules (drugs and therapeutics)

Question 21

Fluorescent polarization

Answer 21

-fluorochrome is placed on the small molecular analyte of interest
-polarized light used as an excitation wavelength
-fluorochrome attached to small molecules will freely rotate and emit light in a variety of directions (depolarize)
-If Ab binds to molecule and fluorochrome, the rotation is slowed and the plan-polarized light remains polarized
-competitive assay: drug in the patient’s sera competes with labeled drug molecules for binding to kit Ab. The more drug in the patient sera the more free-labeled drug and thus less polarized light is detected
-Rapid Rotation = light is depolarized (the more patient sample we add the less polarized the light is)
- Slow Rotation = light is polarized

Question 22

interfering substances

Answer 22

-some inhibiting substances affect many test, like using plasma from EDTA tubes

Question 23

Rheumatoid factor

Answer 23

-Ab to the Fc region of IgG
-false positive for any capture assay

Question 24

Human heterophilic antibodies

Answer 24

-are antibodies to animal Ig present in people who work or are exposed to those animals regularly
-false positive for capture assays (detection Ab of animal origin)

Question 25

Human Anti-Mouse Antibody (HAMA)

Answer 25

-Specific type of human heterophilic Ab that is produced in response to therapeutic or diagnostic mouse monoclonal antibody

Question 26

Enzyme: detection method

Answer 26

-spectrophotometer
-fluorimeter
- luminometer

Question 27

Fluorescent: detection method

Answer 27

fluorimeter

Question 28

radioimmunoassay: detection method

Answer 28

-gamma or scintillation counting

Question 29

chemiluminescence: detection method

Answer 29

luminometer

Question 30

The double capture assay was developed to detect IgM
preferentially over IgG for the diagnosis of a current
infection…. why?

Answer 30

Persistent infections or reinfections will produce IgG antibodies (class switch would have occurred). To diagnose current or acute infections that are new the best way to do would be the detection of IgM.

Question 31

inhibition assay readout

Answer 31

-the antigen concentration is inversely related to the readout