Labeled Antibody Tests

Labels

• fluorescent

• enzyme

• chemiluminescence

• radioactive

DIRECT fluorescent Antibody (DFA) Tests

• Detect Antigen

• detect it in tissue, sample, cell suspension using antigen specific fluorescently labeled Ab 

Conjugate

• what the label or carrier particle is attached to

INDIRECT fluorescent antibody (IFA) Test

• detect unlabeled primary patient antibody in sample using fluorescent labeled secondary antibody

Capture or Sandwich Immunoassay

• detects antigens or antibodies

• can use any label

Indirect Capture

• antibody test to detect an antigen 

Features of Indirect Fluorescent Antibody test

• patient serum and conjugate 

Competitive Immunoassay for antibody

• signal detected is inversely proportional to antibody concentration

Fluorescence- activated cell sorting (FACS)

• flow cytometer 

• identifies antigens on living cells and can sort them 

Fluorescence- activated cell sorting (FACS) Features 

• cells in sample are fluorescently labeled Abs that bind to cell surface molecules

• these are passed and sorted through a LASER in a single file line 

LASER

• light amplified stimulated emitted radiation 

ELSA

• Enzyme-Linked Immunosorbent Assay 

Enzyme labeled reagents

• used to detect antigens and antibodies 

Qualities of Enzyme Labels

• must be stable

• specific

• cannot bind to Ag or Ab independently 

• cannot be inhibited by other parts of reaction mixture

Horseradish peroxidase and alkaline phosphatase

• catalyze oxidation of substrate using H2O2 to form colored product 

• HRP: amber, blue, yellow, green

• ALP: yellow 

Chemiluminescent Labeling

• luminol produces glow of light

• used by crime scene investigators

ELSA

• used for antibody detection

Colorimetry, Fluorimetry, and Chemiluminescence

• amount of patient Ab is measured by assessing amount of end product by optical density scanning of the microtiter plate wells 

Enzyme-Multiplied Immunoassay Technique (EMIT) 

• competitive assay 

• does not need washing step

• easily adapted to automated analyzers

Enzyme-Multiplied Immunoassay Technique Procedure

• incubate patient sample with anti-drug Ab 

• if drug is present it will be bounds by Ab 

• anti-drug Ab remaining binds to enzyme labeled drug and inactivates the enzyme 

• add substrate and detect via colorimeter

Immunochromatographic Test

• lateral flow test 

• capture antibodies immobilized on solid phase 

• rapid tests 

Streptavidin Biotin Labeling

• strong noncovalent interaction

• strepavin has a high affinity for biotin 

• has an amplification of signal 

RAST

• radioallergosorbent test

• measures allergen specific IgE

Western blotting 

• electrophoretically separate subspecies of antigens 

• detect antibodies using labeled anti-human Abs 

• visualize color using chemiluminescence or enzymes 

Dot Blot 

• antigen is added directly to membrane filter and is NOT separated electrophoretically 

Sensitivity 

• ability of a test to correctly identify patients with the disease

Specificity 

• ability of a est to correctly identify patients without the disease

complement Fixation Test

• detect specific antibody 

• complement is used as a regent 

Test System for Complement Fixation Test

• Ag + Ab at unknown concentration in patients serum 

Reagents of Complement Fixation Tests

• sRBCs will lyse in the presence of guinea pig complement 

What happens if hemolysis present (pink) in a complement fixation test?

• test is negative

what if no hemolysis occurs (RBC red pellet) in a complement fixation test?

• test is positive