10. LABELED IMMUNOASSAYS

36問 • 2年前

Yves Laure Pimentel

通報

問題一覧

– labeled Ag is INVERSELY proportional to the ptx Ag Conc.

Competitive Immunoassay

Competitive Immunoassay – labeled Ag is _________ proportional to the ptx Ag Conc.

INVERSELY

- amount of label measured is DIRECTLY proportional to the amount of ptx antigen

Noncompetitive Immunoassay

Noncompetitive Immunoassay - amount of label measured is ___ proportional to the amount of ptx antigen

DIRECTLY

are unlabeled analytes that are made up in known concentrations of the substance to be measured.

Standards/Calibrators

Separation methods a. most immunoassays use a _________-phase vehicle for separation.

SOLID

FIRST immunoassay to apply quantitative

Radioimmunoassay

MOST POPULAR RADIOACTIVE REAGENT

125 IODINE

MEASURES RADIOACTIVITY

Scintillation counter

RIST

(Radioimmunosorbent test)

(PRIST)

Precipitin radioimmunosorbent test

USED FOR TOTAL SERUM IGE

RIST (Radioimmunosorbent test)and Precipitin radioimmunosorbent test (PRIST)

RAST

(Radioallergosorbent test)

USED FOR ALLERGEN SPECIFIC IGE

RAST (Radioallergosorbent test)

MOST COMMON FLUOROPHORE IN Fluorescent Immunoassay

Fluorescein and Rhodamine

Fluorescent Immunoassay Disadvantage

QUENCHING/ DIMINISHING

FITC (?)

Fluorescein Isothiocyanate

MOST COMMON FLUORESCENT LABEL

FITC (Fluorescein Isothiocyanate)

Fluorogenic substrate-label Antigen competing with patient Antigen for antibody. Then unbound leftover labeled Antigen reacts with enzyme to form production. (reaction is directly proportional)

Fluorescent SUBSTRATE -labeled inhibition

Fluorophore labeled Antigen competes with ptx Antigen for antibody. Antibody-bound labelled Antigen ROTATES slowly, emitting lower energy light. (S.2019)

Fluorescent POLARIZATION immunoassay

Oxidation-Reduction -> Light energy

Chemiluminescent Assay

Excellent sensitivity, stable,nontoxic, and inexpensive

Chemiluminescent Assay

Chemiluminescent Assay LEVEL

ATTA MOLE 10-18 ZEPTA MOLE 10-21

No disposal or health prob; one molecule of enzyme can produce multiple products therefore little reagent is needed

Enzyme Immunoassay or ELISA/enzyme-linked immunosorbent assay

have the highest TURN OVER rates, high sensitivity, and are easy to detect, so they are most often used in such assay

ALP and HRP

Antigen Detection: a. Ab on plastic solid phase + Ptx serum (Ag) -> Ag-Ab complex + Enzyme labeled Ab  Sandwiched Antigen (by solid phase Ab and Enzyme labeled Ab) + Chromogenic substrate = Color = concentration

Enzyme Immunoassay or ELISA/enzyme-linked immunosorbent assay

solid phase Ag + Ptx Ab – wash -- > + Labeled AHG – wash -- > + Substrate = Colored Product measured on a SPECIFIED TIME

Noncompetitive enzyme immunoassay

Noncompetitive enzyme immunoassay Test system

SOLID PHASE ANTIGEN

Noncompetitive enzyme immunoassay Indicator system

ENZYME LABELED AHG + SUBSTRATE

Noncompetitive enzyme immunoassay Inadequate 2nd washing results

FALSE INCREASE

Noncompetitive enzyme immunoassay Prolonged (than specified time) reading at the color reaction phase =

FALSE INCREASE

Solid phase Ag + Ptx Ab + Enzyme-labeled Ab specific to solid phase Ag are mixed -> + Chromogenic substrate = Color is inversely proportional to Ab concentration