Clin Chem Exam 3 Immunoassays

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a substance that can elicit an immune response (produce a specific antibody) when injected into a subject (animal or human)

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Antigen (ag)

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

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antibody (ab)

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determines the sensitivity and specificity of an immunoassay

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antibody (ab)

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

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antibody-binding site

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portion of the antigen that combines with the binding site of the antibody

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antigenic determinant

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attraction of a positively charged molecule for a negatively charged molecule

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electrostatic force

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influenced by:
-ionic state of each molecule
-complementary nature of the charges
-distance between molecules

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Electrostatic force

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-electrostatic force
-hydrogen bonding
-hydrophobic force
-van der waals force

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forces that bind antigen to antibody

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-strength of attraction
attraction of two negatively charged atoms for hydrogen
-bond is relatively weak
-occurs best at lower temperatures

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hydrogen bonding

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-the attraction between nonpolar groups
-tends to exclude water
- if immune complex is large it becomes insoluble and precipitates

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hydrophobic force

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weak attractive force between the electron cloud of one atom and the nucleus(protons) of another molecule

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Van der Waals force

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the thermodynamic bond strength of an anitgen-antibody complex

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antibody affinity

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how strongly antibodies bind

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antibody avidity

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

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The law of mass action

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free antibody + free antigen <----> antibody-antigen complex

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The law of mass action

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Involve highly specific and tight, noncovalent, reversible binding between and antigen and its corresponding antibody resulting in the formation of a complex.

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Immunoassays

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depends on the concentration of reactants;avidity, affinity, and specificity of reaction and environmental conditions
-typically useful when the endogenous concentration of an analyte is very low
-analyte of interest may be antigen or antibody

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Immunoassays

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differes from other tests in that reactions are made specific through the use of monoclonal antibodies

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immunoassays

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the same as other tests that we have seen in that light is used to quantify the final product with a photometer

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immunoassays

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the basis for over 70 clinical tests, including tests of therapuetic drugs, hormones, proteins, viruses and their antibodies, and drugs of abuse screening

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Immunoassays

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antibody reacts with antigens that are similar to(but not the same) its homologous antigen

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antibody crossreactivity

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the greater similarity between antigens, the stronger bond between antigen and antibody

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antibody crossreactivity

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-reagent antibodies are produced by injecting animals with the antigen or analytical targets
-antigen and antibody reactions are subject to the law of mass action

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immunoassay techniques

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-polyclonal antibodies may be made directly from animal blood and will bind with a variety of antigens
-monoclonal antibodies are produced as only one type of antibody by dividing to produce a large population of clones all making the same monoclonal antibody

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immunoassay techniques

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

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Immunochemical 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 patients blood)
-inexpensive/economical/ non-toxic
-stable

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desirable properties of immunochemical lables

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-with addition of chromagen, allows the immunoassay result to be quantitated colorimetrically
-Common lables: horseradish peroxidase(HRP), ALP and G6PD

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Enzyme labels

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-detected when a photon is released from a flourescent molecule that is excited from its ground state to a higher state and then returns to ground state
-common labels: flourescein

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Flourescent labels

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-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 intesity is related directly to the concentration of the reactants
-Common labels: luminol, acrydium esters

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Chemiluminescent lables

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radioactive iodine (125I) is most commonly used

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Radioactive labels

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limited reagent assay in which anayte(usually antigen) competes with a labeld antigen for a limited pool of antibody molecules

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Competitive immunoassays

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-labeled antigen concentration is constant and limited
-as analyte (unlabeled Ag) increases, more binds to Ab resulting in less binding of labled Ag.
-Amount of antigen in the sample is inversely related to the amount of label measured in this type of assay

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Competitive immunoassay

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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 or one step competitive assay

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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 equilibriate(incubate)
-after seperation, bound label is determined

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Sequential or two step competitive assay

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-antibody is present in excess and is labeled
-antigen (sample analyte) is bound (sandwiched) between two antibody reagents
-excess ubound labeled antibody is removed by washing and bound label is determined

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Single site non competitive immunoassays

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Concentration of labeled antibody-antigen complex is directly proportional to the concentration of antigen

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single site non competitive immunoassays

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-antigens are attached to a solid phase, such as plastic wells, tubes, capillaries, membranes, latex particles or magnetic particles
-patient 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) non competitive immunoassay

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tends to give falsely low value when serum concentration of the analyte(tumor, marker, hormone, etc.) rises above a certain elevated level

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Hook effect

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Excess patient antigen(prozone) saturates both capture and labeled antibody resulting is almost no complex formation
Solution: routinely dilute the sample and rerun

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Hook effect

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-do not require seperation of bound and free labeled antigen or antibody

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Homogenous immunoassays

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Advantage:
-speed and adaptability
-useful for amall analytes such as drugs
-These assays are competitive

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Homogenous immunoassays

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-require a seperation of bound and free label
-Advantage: sensitivity and specificity
-can be competitive or non-competitive

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Heterogenous immunoassays