Chemistry Instrumentation Solo 1 Lecture 2 Study Guide

What are the three basic components of an immunoassay

Analyte

Antibody

Detectable Label

What is the most commonly used antibody type in immunoassays

IgG

What types of analytes are typically measured using immunoassay

Proteins, hormones, metabolites, therapeutic drugs, and drugs of abuse

What types of labels are used in immunoassays

Horseradish peroxidase

Beta-galactosidase

Alkaline Phosphatase

Name four requirements for enxymes to be used as labels

High amount of stability

Extreme specificity

Absence from the antigen or antibody

No alteration by inhibitor with the system

2 examples of luminescent labels used in chemiluminescence

Luminol

Acridinium Ester

Competitive Immunoassay

Noncompetitive Immunoassay

Which type of assay does not require a physical separation step such as washing?

Homogenous Assay

Which type of assay does require a physical separation step such as washing

Heterogenous Assay

Most common separation technique to distinguish free labeled reactant from bound labeled reactant

Solid Phase

Four formats available for an Enzyme-linked immunoabsorbent assay (ELISA)?

A competitive assay using labeled Ag

A competitive assay using labeled Ab

A noncompetitive assay to detect Ag

A noncompetitive assay to detect Ab

Describe EMIT

Used to test for a drug, hormone, or metabolite in a patient’s sample. An enzyme-labeled drug or molecule is added with patients sample that may or may not contain the same drug or molecule. Antibody specific to the drug, as well as an enzyme substrate, is added. The enzyme-labeled drug/molecule competes with the patient’s drug/molecule for antibody binding sites. The enzyme attached to the drug is affected by antibody binding. When the antibody binds to the enzyme labeled drug, it reduces the enzymes’ ability to catalyze a reaction. The change in enzyme activity can be measured through a change in absorbance detected by the instrument. This change is proportional to the amount of unbound enzyme-labeled drug, which in turn is proportional to the amount of the target substance in the sample.

What is the purpose of a calibration curve

To establish a relationship between the concentration of the analyte and the magnitude of the signal given by the measuring device

What should be done if the signal is below the analytic measurement range

The result is typically reported as less than the lower end of the AMR or less than the lowest calibrator used in the clinical laboratory

What should be done if a signal is reported above the analytical measurement range

The result may be reported as greater than the upper limit of the AMR or greater than the highest calibrator used in the lab. Alternatively, the sample may be diluted to bring the analyte concentration within the AMR and reanalyzed. The measured value on the diluted sample is then multiplied by the dilution factor to determine the concentration in the original sample

2 types of antibodies that may interfere with sanwich immunoassays

Human anti-mouse antibodies

Heterophile antibodies

Immunoassay

An assay based on the reaction of an antibody that is specific for an antigen

Label

A substance with a measurable property attached to an antigen, antibody, or binding substance that will better detect lower concentrations of immune complexes

Stokes Shift

The difference between the excitation wavelength and the emission wavelength

Solid Phase

Solid particles, beads, trays, or tubes onto which antibody or antigen is absorbed

Interpolation

Connecting the points on the calibration plot to form the best fit line or curve; establishes an expected signal for the range of concentrations of analyte that fall between the lowest and highest calibrator

Analytic Measurement Range (AMR)

Also known as the dynamic range, it defines the lowest to highest measurable quantities

Hook Effect

Excess antigen binds to free labeled antibody, prohibiting the labeled antibody to bind to the capture antibody