14 terms
Antigen/Antibody Interactions
Agglutination
Solid or particulate antigen forms a lattice with a soluble antigen
Precipitation
soluble antigen and antibody forms a complex lattice of interlocking aggregates
opsonization
coating of a particle to adhere firmly to the surface of a phagocytic cell to enhance phagocytosis of the target particle
List 5 primary reactions
1) Equilibrium Dialysis
2) Immunofluorescence (Direct and Indirect)
3) Radioimmunoassay
4) RIPA
5) Enzyme linked immunosoerbent assay (ELISA)
2) Immunofluorescence (Direct and Indirect)
3) Radioimmunoassay
4) RIPA
5) Enzyme linked immunosoerbent assay (ELISA)
List 4 Secondary reactions
1) Precipitation
2) Agglutination
3) Complement fixation
4) Nephelometric
2) Agglutination
3) Complement fixation
4) Nephelometric
What is the difference between a primary reaction and a secondary reaction
In a primary reaction only one antibody combining site on the primary secondary and tertiary antibody is occupied. if both sites on any of the antibodies is occupied, then it is a secondary reaction. all secondary reactions involve lattice formation and both antibody and immunogen must be polyvalent.
What is the difference between affinity and avidity
affinity measures the strength of the noncovalent interactions between one antigen binding site and 1 epitope. Avidity measures the combined strength of multiple interactions between a multivalent antibody and antigen.
Direct ELISA vs. Competitive ELISA
In direct ELISA, antibody from serum is added to solid support containing antigen, then enzyme or secondary antibody is added and O.D measured using a standard curve.
In competitive ELISA, antibody from serum is added to support containing antigen, then radiolabeled antibody is added. if radiolabeled antibody binds, it means that original serum did not contain antibody. standar curve also used
In competitive ELISA, antibody from serum is added to support containing antigen, then radiolabeled antibody is added. if radiolabeled antibody binds, it means that original serum did not contain antibody. standar curve also used
How do the standard curves for competitive ELISA vs. Direct ELISA differ?
In direct ELISA, the standard curve has a positive slope, in competitive ELISA, the standard curve has a negative slope. negative slope means that as the serum antibody concentration increases, we will be able to detect less radioactive antibodies.
What does AMINOPTERIN do?
an analog of dihydrofolic acids and binds to dihydrofolate reductase and inhibiting purine synthesis. therefore no DNA.
Hypoxanthine and thymidine in hybridomas
important for the salvage pathway of purine synthesis. Thymidine kinase is important for the utilization of thymidine, HGPRT is important for the utilization of hypoxanthine.
Describe the branched DNA technique
only small sample of DNA needed. DNA complement for target DNA sequence is boud to solid support, target dna added, linker probe added which binds to target DNA and also branched DNA. enzyme is added which causes fluorescence of substrate on branched DNA.
What is Nephelometry
Nephelometry is used in secondary reactions. used to measure the degree of light scatter. the greater the antigen concentration, the greater the degree of light scatter. standardized curve also used here.
Immunodiffusion
can be used to detect the concentration of specific class of immunoglobulin.
1) petridish with semisolid agar filled with antibodies for sayy... IgG
2) cut 5 small holes in solid agar
3) fill 3 holes with known concentrations, fill 2 holes with unknown
4) IgG will diffuse out of hole and precipitate upon meeting with antibody
5) diameter of precipitation corresponds to concentration
6) use known concentrations to make standard curve.
1) petridish with semisolid agar filled with antibodies for sayy... IgG
2) cut 5 small holes in solid agar
3) fill 3 holes with known concentrations, fill 2 holes with unknown
4) IgG will diffuse out of hole and precipitate upon meeting with antibody
5) diameter of precipitation corresponds to concentration
6) use known concentrations to make standard curve.