Immuno Assays Flashcards

Question

Heavy chain

Answer 1

the two chains in the antibody that are longer and have a higher molecular weight

Answer 2

the two chains in the antibody that are shorter and have a lower molecular weight

Answer 3

the upper region of the antibody that contains the Fv, variable region

Answer 4

the bottom region of the antibody that contains the constant regions of the chains

Answer 5

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

Answer 6

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

Answer 7

“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

Answer 8

- 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

Answer 9

- 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)

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

1. Radioisotopes: attach radioactive iodine to Tyr on peptides/proteins; exchange 1H to 3H (tritium); phosphorylate with 32P- containing ATP 2. Fluorescence: fluorophores, ex. fluorescein 3. Whole Enzymes: ex. Horseradish peroxidase or alkaline phosphatase, or G6PDH (glucose 6-phosphate dehydrogenase) 4. Whole Proteins: ex. Green fluorescent protein

Answer 14

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

Answer 15

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

Answer 16

Homogeneous- don’ t have to chemically fix things to the surface - EMIT: homogeneous - FPIA: homogenous - ELISA: heterogeneous - IRMA: heterogeneous - RIA: heterogenous

Answer 17

1. Immunometric 2. Competitive 3. Indirect 4. Immunocapture

Answer 18

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.

Answer 19

- 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

Answer 20

* 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)

Answer 21

- 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.

Answer 22

Coombs test for blood type determination, this uses 2 different species binds to antigen on surface of RBC.

Answer 23

● 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

Immuno Assays Flashcards

(47 cards)