Medchem 423 > Immuno Assays > Flashcards
Immuno Assays Flashcards
Anti-antibody
antibody specific for immunoglobulins
antibody
immunoglobulin with five main types IgA, IgD, IgE, IgG, IgM
Competitive immunoassay
reagent limited immunoassay in which labeled and unlabeled species compete to generate a signal
Conjugate
chemically or physically link two or more distinct molecules
cross-reactivity
binding of an antibody to an antigen
ELISA
Enzyme-linked immunosorbent assay, sandwich enzyme immunoassay; in which one of the reaction components is attached to the surface of a solid phase
to facilitate separation of bound and free labeled reactants.
EMIT
Enzyme-multiplied immunoassay technique; a nonseparation immunoassay using
an enzyme label
Epitope
The specific portion of a macromolecular antigen which an antibody or a T cell receptor binds; an antigenic determinant
FPIA
Fluorescence polarization immunoassay
Hapten
low molecular weight, that is not itself immunogenic, but becomes immunogenic and induces an antibody response after conjugation
Heterogeneous assay
Immunoassay format that uses two phases, usually liquid and solid, to separate reacted/bound from unreacted/unbound components
Homogeneous assay
Labeled-analyte immunoasay format that does not require separation of bound and free antigen
Immunogen
A substance capable of inducing an immune response
Immunoglobulin (Ig)
A protein with antibody activity
Immunometric assay
Immunoassay format using labeled antibody to bind antigen, with two main subdivisions: single-site, and two-site
Interference
Any factor causing bias in an assay result, other than the presence of a true cross-reacting substance
IRMA
Immunoradiometric assay; a type of two-site immunoassay using radioisotopes as
the label on the antibody
Isotope
Atomic species that, in the nucleus, have the same number or protons but a different number of neutrons
Label
Any substance with a measurable property that can be attached to an antigen, antibody, or other binding substance includes chromophores, fluorophores, radioactive isotopes, enzymes
Monoclonal antibody
A monospecific antibody that is produced by a single plasma cell or a single clone of plasma cells
Polyclonal antibodies
A heterogeneous mixture of antibodies with diverse affinities toward an antigen; produced by a large number of plasma cell clones
Radio label
A radioactive label; a label using unstable isotopes that spontaneously transform to a more stable state, emitting energy in the form of particles or electromagnetic
pulses
RIA
Radio-immunoassay; a heterogeneous competition immunoassay that uses radio labeled antigens or antibodies; in the original liquid-phase format, the assay would require a precipitation step to separate antibody-antigen complexes
Western blot
Membrane-based assay in which proteins are separated by electrophoresis, followed by transfer to a membrane and probing with a labeled antibody
Heavy chain
the two chains in the antibody that are longer and have a higher molecular weight
Light chain
the two chains in the antibody that are shorter and have a lower molecular weight
Fab region
the upper region of the antibody that contains the Fv, variable region
Fc region
the bottom region of the antibody that contains the constant regions
of the chains
Sandwich assay
the immunometric assay. When there are two kinds of
antibodies, one labeled and one unlabeled, that are added with analyte in the sample, as the analyte concentration increases, the signal will increase because more analyte in the sample means that more labeled antibody is bound
- Describe the general structure of an immunoglobulin (antibody), noting heavy and light chains, variable and constant amino acid sequence regions, hinge region, and antigen binding sites
It has two heavy chains and two light chains. The heavy chains are the longer chains and the light chains are the shorter chains. The variable region is the Fv region where the antigen will bind to. The Fc part contains the constant amino acid sequence. The hinge region is the region where the chains are joined by the disulfide bonds
- Briefly describe the sources of antibody-antigen binding specificity and affinity. Explain how Ab flexibility, especially with respect to the hinge region, contributes to the crosslinking capabilities of Abs
“Recognition” occurs using weak noncovalent interactions between the epitope on the antigen, and the tips of the arms of the antibody (the CDR).
- Epitope: the specific portion of a macromolecular antigen to which the antibody binds
- The flexible hinge with the disulfide bonds allows the molecule to have more flexibility due to the noncovalent interactions it is easier to manipulate
- The flexibility allows the two Fab regions to bind to two different bacteria or viruses→ crosslinking
- Briefly describe what makes for a good antigen. Given suitable candidates (molecules, cells, etc.) for antigenic behavior, predict which would and which would not likely induce an immune response. Clearly state reasons for your decisions
- High MW, chemical complexity, degradability
- Antigens tend to have high molecular weight. Small compounds themselves are generally not themselves immunogenic. Those with MW > 6000 are generally immunogenic. Chemical complexity: different physico-chemical features. Degradability: capable of being cut into small pieces by certain cells in the immune system (antigen-presenting cells). Proteins generally are “best”- cut by proteases, with lots of diversity in their features. Polysaccharides and nucleic acids – degradable, but not so diverse. Lipids, plastics, individual drug species, etc. – generally NO
- List several advantages of monoclonal antibodies over polyclonal antibodies.
- Identical protein molecules, same Ig class.
- Highly specific, all recognize the same epitope
- Reduced “background” binding (less non-specific or cross-reactive binding)
- Unending supply possible, in large (industrial) volume
Disadvantages:
- Costly
- Time-consuming
- Detects single epitope (both advantage and disadvantage)
- Explain the differences between an immunometric assay and a competitive assay. Given an appropriate sketch or other graphic interpretation, decide whether or not it represented one or the other type of assay. State reasons for your decision.
Competitive assay:
- Used both labeled and unlabeled analyte to combine with the antibody immobilized on a solid phase
- When only labeled and no unlabeled, we should see the strongest signal
- More unlabeled analyte in sample displaces labeled analyte that you add
- less signal because you have less labeled analytes bound to the antibody
- Graph (below): as analyte concentration goes up signal goes down
7 a. Explain the differences between an immunometric assay and a competitive assay. Given an appropriate sketch or other graphic interpretation, decide whether or not it represented one or the other type of assay. State reasons for your decision.
- Competitive Assay
Competitive assay:
- Used both labeled and unlabeled analyte to combine with the antibody immobilized on a solid phase
- When only labeled and no unlabeled, we should see the strongest signal
- More unlabeled analyte in sample displaces labeled analyte that you add
- less signal because you have less labeled analytes bound to the antibody
- Graph (below): as analyte concentration goes up signal goes down
- see page for graph
7 b. Explain the differences between an immunometric assay and a competitive assay. Given an appropriate sketch or other graphic interpretation, decide whether or not it represented one or the other type of assay. State reasons for your decision.
Immunometric assay
Immunometric (sandwich) assay:
- Analyte has two sides, one with pointy end and one with indented end
- 1 antibody is labeled and 1 antibody is not labeled
- Label is on the antibody, not the analyte
- More analyte in the sample means more labeled antibody is bound
- More signal
- Graph (below): as the analyte concentration goes up the signal increases.
- see page for graph
- List the 4 different types of labels used in immunoassays.
- Radioisotopes: attach radioactive iodine to Tyr on peptides/proteins; exchange 1H to 3H (tritium); phosphorylate with 32P- containing ATP
- Fluorescence: fluorophores, ex. fluorescein
- Whole Enzymes: ex. Horseradish peroxidase or alkaline phosphatase, or G6PDH (glucose 6-phosphate dehydrogenase)
- Whole Proteins: ex. Green fluorescent protein
- List common isotopes used in radiolabels that appear in radioimmunoassays.
Explain what and how the signal is detected in these assays.
Note special hazards and precautions needed with radioimmunoassays.
Radioactive labels
• Single atoms (e.g., 1 25I on Tyr)
• Tritium 3 H – weak beta emitter
• Carbon 1 4C – weak beta emitter
• Phosphorus 3 2P – strong beta emitter
• Iodine 1 25I, 1 31I – beta and gamma emitter
• Tridium is a lot safer than iodine. Lower energy and won’t accumulate in the thyroid. Has a long half life.
- detected by bntire functional groups (e.g., methylation with 3 H-SAM)
- No special hazards: relatively uncommon now, due to hassles with radioactivity and improved sensitivity of other formats
- List common enzymes used in those immunoassays employing enzymes; list common substrates for those enzymes.
Explain what and how the signal is detected in these assays.
Enzymes
• Most common are horseradish peroxidase (HRP) and alkaline phosphatase (AP or ALP); also glucose 6-phosphate dehydrogenase (G6PDH)
what • Requires addition of substrate to be detected
how • Conjugation of enzyme with antigen or antibody can affect structure/function, charged particles ionize directly, neutral particles ionize indirectly and penetrate further
- For IRMA, EMIT, FPIA, ELISA, and RIA, note which are homogeneous assays and which are heterogeneous. In the heterogeneous assays, note what is immobilized.
Explain how immobilization can affect the performance of the assay.
Homogeneous- don’ t have to chemically fix things to the surface
- EMIT: homogeneous
- FPIA: homogenous
- ELISA: heterogeneous
- IRMA: heterogeneous
- RIA: heterogenous
- Note the four types of ELISA, and explain their differences. Note particular applications for each type.
- Immunometric
- Competitive
- Indirect
- Immunocapture
- Explain the basis of cross-reactivity in immunoassays.
* Note the effect of cross-reactivity on interpretation of assay results.
* Given suitable information on antigens and antibodies, predict if cross-reactivity is likely.
Cross- reactivity: response by the antibody to substances other than the analyte
- Often happens when analyte has metabolites that are similar in structure, or is a member of a family of structurally-similar compounds (e.g., steroids).
- May also happen with large proteins that have closely related folding patterns or amino acid sequences.
- Net effect is to tie up the antibody on non-analyte compounds, producing interference in the assay.
- List several different types of interference in immunoassays.
* Given suitable details on the type of assay and the suspected interfering agent, predict if the assay signal would be greater than expected or less.
* Explain your reasoning.
- Interference: any factor causing bias in an assay result, other than the presence of a true cross-reacting substance
- Enzyme inhibition: The three enzymes most commonly used in immunoassays are glucose 6-phosphate dehydrogenase (G6PDH), alkaline phosphatase (AP or ALP) and horseradish peroxidase (HRP). All have many residues available for conjugation, without loss of activity
- alkaline phosphatase is inhibited by orthophosphate, zinc chelators (Zn is a necessary cofactor), borate, carbonate, and urea.
- horseradish peroxidase will be inhibited by high concentrations of its substrate hydrogen peroxide. It requires calcium as a cofactor, so will be inhibited by calcium chelators.
- Background signal from chromophore/fluorophore
- NADH and bilirubin fluoresce at longer wavelengths, so appropriate filters should be used in assays monitoring fluorescence.
- Heme absorbs light in the visible and UV; avoid sample contamination with lysed RBCs.
- Several vitamins can absorb in the UV, and some fluoresce strongly (e.g.,
niacin, riboflavin). - Numerous possible causes
- Detection system: endogenous signal-generating substances, enzyme inhibitors, enzyme contamination, catalysts other than desired enzyme
- Problems with separations: incomplete linkage to microtiter plate; poor or labile linkage in conjugates of Ab-enzyme, enzyme-antigen, etc.
- Heterogeneity in Ab preparations (polyclonals especially); variation over batches
- Endogenous Abs that cross-react; also autoantibodies
- Alteration of enzyme, Ab, or Ag conformation during preparation, linkage/conjugation, stabilization, mixing
- Displacement of analyte from physiological binding proteins by other biochemical (e.g., fatty acids displacing thyroid hormones)
- Blockage of analyte due to physiological binding proteins
- Explain how the home tests for pregnancy and fertility work.
- Work by common sandwich format.
- uses monoclonal antibodies; 2 separate antibodies
- Primary AB has dye attached and binds to HCG but is not immoblized
- Secondary Ab binds HCG but is immoblized.
- Ovulation - luteinizing hormone (LH)
- Explain how proteins can be identified by Western blot assays.
- Proteins from a cell lysate, body fluid sample, etc. are denatured by addition of the detergent SDS. SDS coats the protein and unfolds it, forming a micelle whose length is proportional to the length of the polypeptide chain. Nitrocellulose likes to stick to the protein.
- Electrophoresis through a gel (polyacrylamide is common) separates the proteins into bands, according to molecular weight.
- After electrophoresis, the gel strip or slab, containing bands of separated proteins, is layered on wet filter paper, and a sheet of a special polymer or of nitrocellulose paper is layered over both.
- Proteins move with the buffer flow, up onto the nitrocellulose sheet (nitrocellulose binds proteins very well).
- The polyacrylamide gel and the filter paper are removed.
- The sheet is soaked in a “blocking” solution, with a high concentration of protein, e.g., albumin.
- Then it is incubated with a solution of primary antibody (Abs against the suspected Ag protein).
- The blocking solution prevents the Ab from randomly sticking to the sheet.
- Then it is rinsed and incubated with a second Ab solution.
- The secondary Ab is labeled with a fluorescent dye, or with an enzyme, for e.g., an ELISA-type assay, and it is specific for the Fc region of the primary Ab. So the signal appears only for those protein bands binding the primary Ab.
- Explain how blood types are determined by immunoassays.
Coombs test for blood type determination, this uses 2 different species binds to antigen on surface of RBC.
- Describe how antibodies to a pathogen (e.g., HIV or some other virus) may be assayed by a hemagglutination assay.
● We have RBC with HIV antibodies and HIV antibodies with antigens attached
● We get a sandwich and lots of agglutination between the RBC HIV antibodies and the
antibodies attached to antigens
● Monitor if we get clumps and if we do it means that our RBC have antibodies that
recognize the antigen
