Clin Chem Exam 3- Immunoassays

Front 1

is a substance that can elicit an immune response (produce a spicific antibody) when injected into a subject

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Antigen (ag)

Front 2

is an immunoglobulin that is formed in response to a foreign substance (antigen)

Back 2

Antibody (ab)

Front 3

Part of the antibody that makes contact with the antigen during an antigen-antibody reaction

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Antibody-binding site

Front 4

Portion of the antigen that combines with the binding site of the antibody.

Back 4

Antigenic determinant

Front 5

binding is dependent on the rate of diffusion and the probability that collision between two molecules will result in binding.

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The law of mass action

Front 6

involve highly specific and tight, noncovalent, reversible binding between an antigen and its corresponding antibody resulting in the formation of a complex.
reactions are made specific through the use of monoclonal antibodies

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Immunoassays

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A reagent component capable of producing a measurable response that can be attached to an antigen, antibody or binding substance.

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Immunochemical Labels

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Desirable properties of labels:

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Easily attached to antigen/antibody (active)
Easily measured
Does not interfere with antibody/antigen reaction (pure and no interference from the patient’s blood)
Inexpensive / economical / non-toxic
stable

Front 9

With addition of chromagn, allows the immunoassay result to be quantitated colorimetrically.
Common Labels: Horseradish peroxidase (HRP), ALP and G6PD

Back 9

Enzyme labels

Front 10

Detected when a photon is released from a fluorescent molecule that is excited from its ground state to a higher state and then returns to ground state.
Common Labels: fluorescein

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Fluorescent labels

Front 11

Organic compounds become oxidized during the reaction and form an unstable derivative.
Upon return to ground state, they release energy in the form of visible light. The light is measured by a luminometer, and light intensity is related directly to the concentration of the reactants.
Common labels: luminol, acrydium esters

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Chemiluminescent labels

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Radioactive label

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iodine (125I) is most commonly used

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Limited reagent assay in which analyte (usually antigen) competes with a labeled antigen for a limited pool of antibody molecules.
Amount of antigen in the sample is inversely related to the amount of label measured in the competitive immunoassay.

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Competitive Immunoassays

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All reactants are mixed simultaneously (one step)
L-Ag and unlabeled Ag (sample) compete to bind with antibody
Bound labeled antigen is inversely proportional to concentration of unlabeled antigen

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Simultaneous (one step), Competitive

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Unlabeled antigen (sample) is first mixed with antibody (in excess) and binding is allowed to reach equilibrium.
Labeled antigen is then added sequentially and allowed to equilibrate (incubate)
After separation, bound label is determined.

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Sequential (two step), Competitive

Front 16

Antibody is present in excess and is labeled.
Antigen (sample analyte) is bound (sandwiched) between two antibody reagents.
As antigen (sample analyte) increases, amount of labeled antibody-antigen complex also increases.
Concentration of labeled antibody-antigen complex is directly proportional to the concentration of antigen.

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Single site
Noncompetitive immunoassays

Front 17

Antigens are attached to a solid phase, such as plastic wells, tubes, capillaries, membranes, latex particles or magnetic particles
Patient antibody is then added to the test system which binds antigen and excess is washed away.
A second antibody that is tagged with a label is then introduced into the test system.
Excess labeled antibody is washed away. The label bound is measured. The amount of label is directly proportional to the amount of antibody present in the patient sample.

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Two site (sandwich)
Noncompetitive immunoassays

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Tends to give a falsely low value when serum concentration of the analyte (tumor marker, hormone etc.) rises above a certain elevated level.
Solution: Routinely dilute the sample and rerun

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Hook Effect

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How to fix Antigen Excess

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Too much antigen so reagent gets overwhelmed > false decreased
Dilute and rerun

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Do not require separation of bound and free labeled antigen or antibody
Advantage:
-Speed and adaptability
-Useful for small analytes such as drugs
All homogeneous assays are competitive

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Homogeneous immunoassays

Front 21

They require separation of bound and free label
Advantage: Sensitivity and specificity
Can be competitive or non-competitive

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Heterogeneous immunoassays

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other Immunoassays

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ELISA
EMIT
MEIA
FPIA