First Aid, Chapter 4 Laboratory Tests, Immunoglobulin Measurement Flashcards
What are the three zones of antibody-antigen interactions? What is the ratio in each zone and how many antigens bound per antibody?
- Zone of Antibody Excess: Complexes exist as single antibody to single antigen. -Zone of Equivalence: Single antibody bind to two antigens, forming large insoluble lattices (i.e., can increase turbidity of solution, appear as precipitin lines).
- Zone of Antigen Excess: Binding sites on the antibodies are saturated, and antibodies exist as single antibody to two antigens.
What is used to measure antibody excess?
Immunoblotting Enzyme-Linked Immunosorbent Assay (ELISA)
What is used to measure antigen-antibody equivalence?
Radial Immunodiffusion (RID) Double Immunodiffusion (Ouchterlony) Nephelometry
What is used to measure antigen excess?
Radioimmunoassay (RIA) ELISA
How are serum immunoglobulins measured?
Nephelometry
What is nephelometry? What quantities of protein are measured?
Nephelometry is a method by which the turbidity of a solution is determined by a pattern of scattered light (Figure 4-2). In the determination of Ig levels, Ig are injected into a reaction chamber with antigen-specific antibody. As antibody-antigen complexes form, light is applied and the extent of light scatter is measured by a nephelometer. Photons are reflected from the immune complexes at an angle and measured with a photomultiplier tube.The amount of light detected is quantified as a function of time and the concentrations of antibody and antigen. This automated technique allows laboratories to process a high volume of samples. Nephelometry is considered to be more sensitive than RID and can measure quantities of protein from 1–10 µg/mL.
Describe how radial immunodiffusion (RID) works. Is it qualitative or quantitative? What quantities can be measured.
Antiserum is mixed into a warm gel matrix while it is still in liquid form, then poured into a flat plate and allowed to cool. Antigen (IgG) is then placed into wells that have been cut into the gel and diffuses into the gel matrix. At the zone of equivalence, a precipitin line is seen (Figure 43). The amount of antigen is determined by the diameter of the precipitin ring, which is compared with a standard curve, created by known serially diluted concentrations of the antigen (see Figure 4-3). This is a quantitative method. Limitations include the inability to accurately measure serum protein concentrations less than 10 µg/mL.
What are advantages of Laurell Rocket over RID? What methods does it incorporate? How much time difference is there between this method and RID?
The Laurell rocket method is considered to be less timeconsuming and more accurate than RID. This method incorporates features of immunoelectrophoresis and the immunodiffusion technique. The addition of immunoelectrophoresis decreases the time involved considerably, from up to 48 hours in RID to several minutes for the Laurell rocket technique.
Describe how the Laurell Rocket method works. Is it qualitative or quantitative?
Samples are added to gel plates containing antiserum in holes made throughout the gel layer. A voltage is applied for a predetermined length of time. A precipitin line is seen at the zone of equivalence, which adopts the shape of a “rocket” (Figure 4-4). The height of the precipitin line is used to determine the amount of antigen concentration, compared against a standard curve. This represents both a qualitative and quantitative method of the antigen placed in the gel.
What is the method of choice to determine immunoglobulin levels?
Nephelometry.
Which methods of determining immunoglobulin levels are insensitive?
RID and Laurell Rocket.
What are the methods of characterizing immunoglobulins?
- Immunodiffusion (Ouchterlony)
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Western Blot (Protein Immunoblot)
- Immunofixation Electrophoresis (IFE)
Describe immunodeiffusion (Ouchterlony). What is the Wadsworth method and how long does it take?
The Ouchterlony method utilizes a doubleimmunodiffusion (DID) technique. Holes are punched into an agarose gel. Antigens are placed in wells, and serum is placed in adjacent wells. Antigen and antibodies diffuse into the gel, and complexes form at the zone of equivalence, which is seen as a white precipitin line (Figure 4-5). The Wadsworth method simplifies the Ouchterlony method and utilizes agar on glass slides. Diffusion occurs within hours and requires fewer reagents.
Which test can be used to detect both excess antibody and excess antigen?
ELISA
How does ELISA work? What types of labels are used? How is the signal detected?
ELISA refers to assays performed on a microtiter plate in either a zone of antigen excess or zone of antibody excess. Antigens and reagents are immobilized by adsorption onto microtiter plates. ELISAs can be performed in many different ways by modifying the basic principle of identifying the bound protein by an antibody, which is conjugated to a label (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, streptavidin, and fluorescent tags). Figure 4-6 illustrates common ELISA formats. The appropriate substrate is added, and the signal is detected by use of a spectrophotometer, fluorometer, or luminometer.
