Module 13: Immunoassays

Question 1

method of immunoassay

Answer 1

antigen-antibody reactions to identify and quantitate analyte in clinical ssamples
Labels often attached to regain components to produce a measurable signal

Question 2

how sensitive is immunoassay

Answer 2

high levels of specificity and sensitivity

Question 3

analytes commonly measured by immunoassay

Answer 3

hormones
therapeutic drugs
vitamins
serological markers (hepatitis antibodies)
metabolites (folate, ferritin)
cancer markers (CA 125, PSA)
cardiac markers (CK-MB, troponin)

Question 4

label

Answer 4

a reagent component that produces a measurable signal

Question 5

capture antibody

Answer 5

reagent antibody designed to capture the analyte of interest

Question 6

conjugate

Answer 6

a reagent antibody with an attached label

Question 7

tracer

Answer 7

a reagent antigen with an attached label

Question 8

radioactive isotopes

Answer 8

labels involve attaching radioactive isotopes to either reagent antigens or antibodies, often isotopes of iodine or cobalt

Question 9

Fluorescent

Answer 9

labels are fluorescent substances (flurophores) that will be excited by certain short, high energy wavelengths of light and then emit longer, lower energy wavelengths

Question 10

Enzymes and substrates

Answer 10

enzyme itself does not produce a signal

Substrate added to reaction, which the enzymes catalyzes into a specific product

Question 11

Chemiluminesscent

Answer 11

labels emit light as a result of chemical reaction
chemilunimescent component attached to reagent component and often a trigger is added to start the chemical reaction
Best sensitivity among immunoassay methods

Question 12

competitive

Answer 12

called Limited reagent
only a low, fixed quantity of reagent capture antibody is added and only one antibody is used
Labelled tracer competes with analyte in sample for limited binding sites
Indirect relationship between analyte and signal produced

Question 13

Non competitive

Answer 13

2 reagent antibodies
One is the capture antibody
Second is conjugate antibody
Often a direct relationship
"sandwich assay"

Question 14

Heterogeneous

Answer 14

assays that use labels that produce the same signal whether bound or not require a separation step
End with 2 solution

Question 15

Homogeneous

Answer 15

assays that employ labels that will produce different signals when bound versus when free do not require a separation step
End with 1 solution

Question 16

Simultaneous methods

Answer 16

all the reagent and sample are added together at once (one step)

Question 17

Sequential methods

Answer 17

sample is added first to interact with reagent capture antibody, allowed to incubate, and then the competing reagent labelled antigen is added (two step)

Question 18

Radioimmunoassays (RIA)

Answer 18

due to safety concerns of use of radioactive materials, is not common

Question 19

Immunoradiometric assay (IRMA)

Answer 19

radioactive labelled antibody was used instead of labelled antigen

Question 20

Enzyme immunoassays (EIA)

Answer 20

use enzyme labelling system to produce measurable signal

Ex. ELISA

Question 21

Cloned Enzyme Donor Immunoassay (CEDIA)

Answer 21

once an enzyme-labelled antigen is bound to an antibody, the enzyme fragment cannot combine with the other half of the enzyme to form an active intact enzyme

Advantage of not requiring separation step but $$$$

Question 22

Fluorescent Polarization immunoassay (FPIA)

Answer 22

uses fluorescent labelled antigen
when exposed to polarized excitation light, the bound fluorescent tracer will emit light that is still polarized int he same plane as the excitation light

Question 23

High dose effect

Answer 23

if there are very high levels of analyte in sample, all capture binding sites may become saturated and excess analyte will remain in solution. Excess may be washed away during separation step and not measured

Results in falsely decreased result
Termed “High dose hook effect” due to apparent of obtained graph of analyte concentration vs signal

Dilute sample and retest to avoid this

Question 24

Heterophile Antibodies

Answer 24

antibodies produced against the antibodies of other species

If patient has developed these to the specific species used as a source of reagent antibody, there can be interference

Question 25

Common heterophile antibody

Answer 25

human anti-mouse antibody (HAMA)

Question 26

Techniques used to help identify or account for heterophiles antibody interferences

Answer 26

repeat testing using different method
repeat test using different sample type (serum vs urine)
running serial dilutions of sample to determine linear response
Use of blocking agents designed specifically to address known interferences