Immunoassays & Microscopy

Question 1

RIA uses labels known as ___. What are the two types?

Answer 1

Radiolabels - iodine 125

Competitive RIA = antigen is labeled; tracer molecule
Non-competitive = antibody is labeled

Question 2

RIA - drawback

Answer 2

Hazardous, short half-life, expensive to dispose

Question 3

Common enzyme labels (3)

Answer 3

Horseradish peroxidase

Alkaline phosphatase

Glucose-6-PD

Question 4

How enzyme labels are measured

Answer 4

Enzyme reacts with substrate to produce a color change

-darker color = higher intensity

Question 5

Common enzyme substrate

Answer 5

Orthophenylene diamine (OPD)

Question 6

Common fluorescent labels and the colors they emit (2)

Answer 6

FITC = green

Rhodamine = orange

Question 7

Common chemiluminescent labels (3)

Answer 7

Acridinium esters

Luminol

Nitrophenyl oxalates

Question 8

Describe a competitive immunoassay

Answer 8

Labeled antigens are mixed with a patient’s antigens and competes for binding sites on a limited amount of antibodies. Measure bound labeled signal. Higher signals indicate more labeled antigens have bound to the antibodies

• lower signal = higher concentration of patient antibodies bound
• higher signal = lower concentration of patient antibodies bound
• inversely proportional

Question 9

Describe a non-competitive immunoassay

Answer 9

“Sandwich” assays

• antigen or antibody sandwiched in the middle
• concentration directly proportional to signal

Question 10

Difference between competitive vs non-competitive immunoassays as they relate to the signal given. Which one is more sensitive and specific?

Answer 10

Competitive = signal inversely proportional to concentration

Non-competitive = signal proportional to concentration
-more sensitive and specific

Question 11

Homogeneous vs heterogeneous immunoassay. How are they different? Which one is more sensitive?

Answer 11

Homogeneous - requires no washing to remove unbound complexes. Antigen-antibody measured directly
-latex agglutination

Heterogeneous - requires washing

• removes background signal from unbound
• less false positive
• more sensitive

Question 12

EMIT - commonly used for…

Answer 12

Drug testing

-mnemonic - EMIT acquit

Question 13

Some components of pre-analytical, analytical, and post-analytical to consider when doing an immunoassay

Answer 13

Pre-analytical example = draw time and detection of Hepatitis Antibody to core, surface or envelope antigens

Analytical = have to do right test, right reagent, right kit to detect marker, right enzymes, right substrates, controls, QC

Post-analytical = ELISA result, confirmatory testing
-presumptive reporting when patient needs immediate results

Question 14

Which immunoassay is most subjective and requires interpretation by 2 CLSs?

Answer 14

Fluorescent techniques
ANA
FTA-ABS
FPIA

Question 15

Nephelometry - what is it used to detect? Based on what principle?

Answer 15

Ig quantitation, antigen-antibody complexes

Light scatter/reflected back
-more scatter = more concentrated sample (more complex)

Question 16

Fluorescent microscopy - the excitation light that hits the specimen is called… The light that is emitted from the specimen is called…

Answer 16

Incident light - hits specimen, excites electron, releases emission light

Fluorescence

Question 17

Incident vs fluorescence light - which wavelength is smaller or higher? Which wavelength has higher energy?

Answer 17

Incident = smaller = higher energy
-495 nm

Fluorescence = higher
-520 nm

Question 18

Labeled fluorescent antibodies are called

Answer 18

Fluorophores or fluorochromes

Question 19

The ability of specimens to absorb and re-radiate light is called… How do fluorescent microscopes deal with this?

Answer 19

Photoluminescence

Separate weaker fluorescent signal from brighter excitation lights

Question 20

What is epifluorescence

Answer 20

Combination of excitation and emission wavelengths travel through specimen, emitting fluorescence
-objective lens both release excitation light and capture emission light