It is determined by serially diluting patient's serum and adding the antigen to see which dilutions give a positive reaction. The highest dilution of serum is the titer, which is expressed as the reciprocal of dilution.
Describe the method used to determine the titer of antibody in a patient's serum.
Hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions.
What are the noncovalent interactions that form the basis of antigen-antibody binding?
The combined strength of the noncovalent interactions between a single Ag-binding site on an Ab and a single epitope.
The strength of antigen-antibody binding when multiple epitopes on an antigen interact with multiple binding sites of an antibody
High excess in antibody concentration relative to antigen concentration.
Describe the circumstance under which soluble immune complexes are produced.
AIDS uses the indirect technique, whereas hepatitis testing involves the sandwich technique.
What is the basic difference between testing for AIDS (HIV) and hepatitis (HBsAg) by ELISA?
Fluorescein, rhodamine, and phycoerythrin
What are the reagents involved in immunofluorescence?
Organic dye that absorbs blue light and emits yellow-green fluorescence.
Organic dye that absorbs yellow-green range and emits deep red fluorescence. Can be used in 2 color immunofluorescence.
Absorber of light and brilliant emitter of red fluorescence.
fluorochromes, Fc region
In immunofluorescence, ____ are conjugated to the ____ of an antibody without affecting the specificity of the antibody. Antibody molecules bound to antigen in cells or tissues will emit light at a characteristic wavelength.
Method of immunofluorescence in which the primary antibody is directly conjugated with fluorescein.
Method of immunofluorescence in which primary antibody is unlabeled and detected with additional fluorochrome-labeled reagent.
First, urine or blood sample is incubated with antibody specific for suspected drug. Antigen-coated particles are then added, and if the particles are not agglutinated by the antibody present, this means that the sample contained an antigen recognized by the antibody, suggesting that the individual was using the drug.
Describe the sequence of steps involved in hemagglutination inhibition.
antigen; primary antibody; enzyme-conjugated antibody; enzyme substrate
In an indirect ELISA, ____ is coated to the titer and washed, after which ____ is added to it, allowing them to react. It is washed, and ____ is added, which binds the primary antibody. Next, it is washed again, and ____ is added to detect whether or not there is an antibody-antigen complex. Finally, using a specialized spectrophotometric plate reader, color is determined. Color means that there was a complex.
primary antibody; antigen; enzyme-conjugated antibody; enzyme substrate
In a sandwich ELISA, ____ is coated to the titer and washed, after which ____ is added to it, allowing them to interact. It is washed again, and ____ is added to it, which binds a different epitope on the antigen. Finally, it is washed again and a ____ is added to detect the complex.
SDS; SDS polyacrylamide gell (SDS-PAGE); nitrocellulose or nylon; enzyme-linked antibodies; ELISA reaction
In Western blotting, a protein mixture is first treated with ____, a strong denaturing detergent. Then, it is separated by electrophoresis in an ____, which separates the components according to molecular weight. The gell is removed from the apparatus and applied to a protein-binding sheet of ____ or ____, and the proteins in the gel are transferred to the sheet by the passage of an electric current. The antigens of interest are detected by addition of ____, and the position of the antibodies is visualized by means of an ____.
A laser beam and light detector count single cells in suspension. Every time a cell passes beam, light is deflected from detector and count is recorded. Fluorescent-tagged cells are excited by the laser and emit light that is recorded by a second detector system. Its deflection plates allow the separation of cell types.
Describe the sequence of steps involved in flow cytometry.