Any immunoassay that uses an enzyme as label.

A substrate is added to measure enzyme activity.

Classifications of EIA

Classifications of EIA

ALP, Horseradish Peroxidase, Labels D-galactosidase, G6PD

Detection Enzymes react with substrate to produce color change

Advantages: Sensitivity. Specificity. No health hazard Advantages No disposal problems. Reagents with long shelf life. Can be automated.

Disadvantages: Natural inhibitors in some specimens. Nonspecific protein binding.

Enzyme-labeled antigen & unlabeled patient antigen compete for binding sites on antibody attached to solid phase. Free labeled antigen is removed by washing. Substrate is added. Color inversely proportional to concentration of analyte in specimen.

Used to measure small relatively pure Application antigens (e.g, drugs, insulin, estrogen)

Ag attached to solid phase. Ab in specimen attaches. Unbound ab removed by washing. Enzyme-labeled antiglobulin added. Attaches to ab on solid phase. Substrate added. Color directly proportional to ab concentration.

Used to detect abs to viruses, e.g., HIV, HAV, HCV, EBV.

Ab attached to solid phase. Ag in specimen attaches Enzyme labeled ab added, attaches to different determinant Enzymatic activity directly proportional to amount of ag in sample.

Used to measure Igs, hormones, proteins & detect tumor markers, viruses, parasites, fungi.

Less sensitive than heterogeneous assays but rapid and simple to perform

Easily adapted to automation. No need for washing or separation steps. Suitable for low-molecular-weight analytes like hormones, therapeutic drugs, and drugs of abuse in serum and urine.

- Based on changes in enzyme activity when antigen-antibody binding occurs - The enzyme tag on the reagent antigen is blocked upon antibody binding, resulting in a loss of enzyme activity - It's a competitive assay where free analyte competes with enzyme-labeled analyte for binding sites

Are frequently enzyme used in homogeneous assays

Achieves similar sensitivity to radioimmunoassay (RIA) without health hazards or disposal issues.

No expensive instrumentation required-can be read using spectrophotometry or simple color observation. Inexpensive reagents with a long shelf life.

Some specimens may contain natural inhibitors. The size of the enzyme label can limit assay design. Nonspecific protein binding may affect results.

The analyte (the substance being tested, like a drug or hormone) competes with an enzyme-labeled antigen for binding to a specific antibody

When the concentration of the analyte in the sample is high, less of the enzyme-labeled antigen binds to the antibody, resulting in?

When the analyte concentration is low, more enzyme-labeled antigen binds resulting in higher enzyme activity?

Which is true?

Membrane-based and easy to perform. Provide reproducible results.

Primarily designed for point-of-care or home testing, but many have been modified for increased sensitivity and semiquantitative use in clinical laboratories.

Typically single-use, disposable assays in plastic cartridges.

Usually made of ___________ that easily immobilizes proteins.

The rapid flow through the membrane and its large surface area enhance the speed and sensitivity of the reaction

Either antigen or antibody can be coupled to the membrane. The reaction is read by detecting the colored product formed.

Types of Rapid Assays:

Rapid Immunoassay uses what type of chromatography?

Analyte is applied at one end of the strip, migrating toward the detection zone. Absorbent pad maintains a constant capillary flow rate, Colored line forms in the detection zone if the immune complex is captured, indicating a positive test

Immunochromatography

Used for microorganism detection (e.g, Streptococcus pyogenes for strep throat).

Used for pregnancy testing and detecting cardiac troponin after a heart attack

Results are typically qualitative (positive or negative)

After addition of sample and proper incubation, a pregnancy test kit without line in the "Control region" is interpreted as?

After addition of sample and proper incubation, a pregnancy test kit with line in the "Test region" is interpreted as?

After addition of sample and proper incubation, a pregnancy test kit with line in the "Control and Test region" is interpreted as?

Demonstrated that antibodies could be labeled fluorescent molecules (fluorophores or fluorochromes)

In 1941, Albert Coons demonstrated that antibodies could be labeled with fluorescent molecules (fluorophores or fluorochromes)

A type of fluorescent molecules: Green

A type of fluorescent molecules: Orange

490-495 nm

550 nm

Absorb energy from an incident light source and convert it into light of a longer wavelength and lower energy when excited electrons return to the ground state.

Ideal Fluorescent Probe Properties:

Solid-phase antigen + Labeled antibody = Antigen-antibody combination fluorescence

Solid-phase antigen + Unlabeled antibody = Antigen-antibody combination + Labeled anti-immunoglobulin = Fluorescence

Based on the change in polarization of fluorescent light emitted by a labeled molecule when bound by antibody

The molecule's rotation rate determines whetner light is polarized or unpolarized.

More antigen in the sample leads to less polarization and vice versa, making fluorescence polarization inversely proportional to the analyte concentration.

Which is true about FLUORESCENCE POLARIZATION IMMUNOASSAY?

Primarily used for measuring low molecular weight analytes such as therapeutic drugs and hormones. Requires sophisticated instrumentation, making it suitable for automated analyzers.

Specimen on glass slide overlaid with fluorescein-labeled ab. If corresponding ag present, fabeled ab binds. Fluorescence observed with fluorescent microscope.

Reagent ag on glass slide overlaid with patient serum. If corresponding ab present in serum, attaches to aq, When fluorescein-labeled antihuman globulin added, attaches to ab. Fluorescence observed with fluorescent microscope

Labeled ag competes with ag in specimen for sites on reagent ab. Free labeled ag rotates rapidly, emits little polarized light. Bound labeled ag rotates more slowiy, emits more polarized light. Amount of polarized light is inversely proportional to concentration of ag in specimen.

Detects ags. Fluorescent labels: fluorescein isothiocyanate or rhodamine B sothiocyanate.

"Sandwich technique." Detects abs in serum.

Competitive. Homogeneous. Automated.

Analytes: Bacterial, viral antigens

Analytes: Fluorescent antinuclear antibody (FANA), fluorescent treponemal antibody (FTA)

Analytes: Therapeutic drugs, hormones

The emission of light caused by a chemical reaction

Oxidation

Common substances used for chemiluminescence include luminol, acridinium esters, ruthenium derivatives, and nitrophenyl oxalates.

Light emissions can range from quick fiashes to continuous grows depending on the substance.

Which is true regarding CHEMILUMINESCENT IMMUNOASSAYS (CLIA)?

Involves electrochemical compounds that generate light through an oxidation-reduction reaction

Ruthenium undergoes a reaction with tripropylamine (TPA) at the surface of an electrode, emitting measurable light.

Often used with magnetic beads to capture the labeled antibody.

Which electrochemiluminescence immunoassay?

Potential for the Future: Assays are gaining wider application in immunologic testing and have great potential moving forward

Labels: Luminol,acridium esters, ruthenium derivatives, nitrophenyl oxalates

Detection: Molecules produce light from chemical reaction.

Type(s) of Assays: Competitive & noncompetitive. Heterogeneous & homogeneous.

Advantages: Same with EIA & IFA

Disadvantages: Quenching of light emission by some biological materials